U.S. patent application number 12/211073 was filed with the patent office on 2010-08-19 for regulation of function of angiopoietin.
Invention is credited to Qin Yu.
Application Number | 20100210520 12/211073 |
Document ID | / |
Family ID | 39764160 |
Filed Date | 2010-08-19 |
United States Patent
Application |
20100210520 |
Kind Code |
A9 |
Yu; Qin |
August 19, 2010 |
REGULATION OF FUNCTION OF ANGIOPOIETIN
Abstract
Pharmaceutical compositions that comprise a pharmaceutically
acceptable carrier and either a therapeutically effective amount of
an ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1
and/or SEQ ID NO:2 or a homologous peptide thereof and
pharmaceutical compositions that comprise a pharmaceutically
acceptable carrier and a vector comprising a nucleic acid molecule
that comprises the nucleotide sequence that encodes an ECM-binding
fragment of Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID
NO:2 or a homologous peptide thereof are disclosed. Methods of
using such compositions to treat individuals suspected of having
coronary artery disease, vascular disease or a condition involving
ischemia and to promote angiogenesis, endothelial survival and
maintaining vascular integrity are disclosed. Methods to identify
compounds that modulates binding of Ang-1 to ECM are disclosed.
Pharmaceutical compositions which comprise a therapeutically
effective amount of Ang-2 protein and/or a vector comprising a
nucleic acid molecule that comprises the nucleotide coding sequence
of Ang-2 and methods of using such compositions to treat
individuals suspected of having cancer are disclosed.
Inventors: |
Yu; Qin; (Philadelphia,
PA) |
Correspondence
Address: |
Pepper Hamilton LLP
400 Berwyn Park, 899 Cassatt Road
Berwyn
PA
19312-1183
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20090209462 A1 |
August 20, 2009 |
|
|
Family ID: |
39764160 |
Appl. No.: |
12/211073 |
Filed: |
September 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10375765 |
Feb 26, 2003 |
7427594 |
|
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12211073 |
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60359679 |
Feb 26, 2002 |
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Current U.S.
Class: |
514/1.1 ;
424/93.2; 435/7.1; 514/44R |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/515 20130101 |
Class at
Publication: |
514/12 ; 435/7.1;
514/44.R; 424/93.2 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61K 31/7088 20060101 A61K031/7088; G01N 33/53 20060101
G01N033/53 |
Claims
1. A pharmaceutical composition comprising a) a pharmaceutically
acceptable carrier and b) a therapeutically effective amount of a
vector comprising a nucleic acid molecule that comprises the
nucleotide sequence that encodes an ECM-binding fragment of Ang-1
protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 or a
homologous peptide thereof.
2. The pharmaceutical composition of claim 1 further comprising a
therapeutically effective amount of an ECM-binding fragment of
Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 or a
homologous peptide thereof.
3. The pharmaceutical composition of claim 1 comprising a vector
comprising a nucleic acid molecule that comprises the nucleotide
sequence that encodes an ECM-binding fragment of Ang-1 protein that
comprises SEQ ID NO:1 and/or SEQ ID NO:2.
4. The pharmaceutical composition of claim 3 comprising wherein
said vector is a viral vector.
5. The pharmaceutical composition of claim 3 comprising wherein
said vector is a DNA plasmid.
6. A method of treating an individual suspected of having coronary
artery disease, vascular disease or a condition involving ischemia
comprising the step of administering to said individual a
pharmaceutical composition comprising a) a pharmaceutically
acceptable carrier and b) therapeutically effective amount of a
vector comprising a nucleic acid molecule that comprises the
nucleotide sequence that encodes an ECM-binding fragment of Ang-1
protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 or a
homologous peptide thereof.
7. The method of claim 6 wherein said pharmaceutical composition
further comprises a therapeutically effective amount of an
ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1
and/or SEQ ID NO:2 or a homologous peptide thereof.
8. The method of claim 6 wherein said pharmaceutical composition
comprises a vector comprising a nucleic acid molecule that
comprises the nucleotide sequence that encodes an ECM-binding
fragment of Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID
NO:2.
9. A method of promoting angiogenesis, endothelial survival and
maintaining vascular integrity in an individual comprising the step
of administering to said individual a pharmaceutical composition
comprising a) a pharmaceutically acceptable carrier and b)
therapeutically effective amount of a vector comprising a nucleic
acid molecule that comprises the nucleotide sequence that encodes
an ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1
and/or SEQ ID NO:2 or a homologous peptide thereof.
10. The method of claim 9 wherein said pharmaceutical composition
further comprises a therapeutically effective amount of an
ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1
and/or SEQ ID NO:2 or a homologous peptide thereof.
11. The method of claim 9 wherein said pharmaceutical composition
comprises a vector comprising a nucleic acid molecule that
comprises the nucleotide sequence that encodes an ECM-binding
fragment of Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID
NO:2.
12. A method of identifying compounds that modulates binding of
Ang-1 to ECM comprising performing a test assay that comprises the
steps of: a) contacting a protein that comprises at least an
ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1
and/or SEQ ID NO:2 with ECM material in the presence of a test
compound; b) measuring the level of binding of said protein that
comprises at least an ECM-binding fragment of Ang-1 protein that
comprises SEQ ID NO:1 and/or SEQ ID NO:2 with said ECM; and c)
comparing said level with the level of binding of protein that
comprises at least an ECM-binding fragment of Ang-1 protein that
comprises SEQ ID NO: and/or SEQ ID NO:2 with ECM material in the
absence of said test compound; wherein when the level of binding of
said protein that comprises at least an ECM-binding fragment of
Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with
said ECM in the presence of said test compound is less than the
level of binding of said protein that comprises at least an
ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1
and/or SEQ ID NO:2 with said ECM in the absence of said test
compound results indicate that the test compound modulates binding
of Ang-1 to ECM by inhibiting said binding and when the level of
binding of said protein that comprises at least an ECM-binding
fragment of Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID
NO:2 with said ECM in the presence of said test compound is more
than the level of binding of said protein that comprises at least
an ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1
and/or SEQ ID NO:2 with said ECM in the absence of said test
compound results indicate that the test compound modulates binding
of Ang-1 to ECM by enhancing said binding.
13. The method of claim 12 wherein the protein is Ang-1
protein.
14. The method of claim 12 wherein the protein is a fragment of
Ang-1 protein.
15. The method of claim 12 wherein the protein comprises a
detectable label.
16. The method of claim 15 wherein the level of binding is measured
by measuring the amount of detectable label present on said ECM
after removing unbound protein from said ECM material.
17. The method of claim 12 wherein the ECM material is produced by
culturing cells on a substrate for a sufficient time for said cells
to produce ECM material on said substrate and removing said cells
from said substrate without removing said ECM material.
18. The method of claim 17 wherein said cells are Lewis lung
carcinoma cells or TA3 murine mammary carcinoma cells.
19. The method of claim 12 wherein said method comprising multiple
test assays which are identical except that the amount of test
compound used differs.
20. The method of claim 12 the level of binding of protein that
comprises at least an ECM-binding fragment of Ang-1 protein that
comprises SEQ ID NO; I and/or SEQ ID NO:2 with ECM material in the
absence of said test compound is determined by performing a control
assay wherein said control assay comprises the steps of a)
contacting a protein that comprises at least an ECM-binding
fragment of Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID
NO:2 with ECM material in the absence of a test compound; b)
measuring the level of binding of said protein that comprises at
least an ECM-binding fragment of Ang-1 protein that comprises SEQ
ED NO:1 and/or SEQ ID NO:2 with said ECM.
21. A pharmaceutical composition comprising a) a pharmaceutically
acceptable carrier and b) a therapeutically effective amount of
Ang-2 protein and/or a vector comprising a nucleic acid molecule
that comprises the nucleotide coding sequence of Ang-2.
22. The pharmaceutical composition of claim 21 comprising a
therapeutically effective amount of Ang-2 protein.
23. The pharmaceutical composition of claim 21 comprising a vector
comprising a nucleic acid molecule that comprises the nucleotide
coding sequence of Ang-2.
24. The pharmaceutical composition of claim 23 comprising wherein
said vector is a viral vector.
25. The pharmaceutical composition of claim 23 comprising wherein
said vector is a DNA plasmid.
26. A method of treating an individual suspected of having cancer
comprising the step of administering to said individual a
pharmaceutical composition comprising a) a pharmaceutically
acceptable carrier and b) a therapeutically effective amount of
Ang-2 protein and/or a vector comprising a nucleic acid molecule
that comprises the nucleotide coding sequence of Ang-2.
27. The method of claim 26 wherein said pharmaceutical composition
comprises a therapeutically effective amount of Ang-2 protein.
28. The method of claim 26 wherein said pharmaceutical composition
comprises a vector comprising a nucleic acid molecule that
comprises the nucleotide coding sequence of Ang-2.
29. The method of claim 28 wherein said vector is a viral
vector.
30. The method of claim 28 wherein said vector is a DNA plasmid.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Ser.
No. 60/359,679 filed Feb. 26, 2002, which is incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates activities of Angiopoietin-1
(Ang-1) and Angiopoietin-2 (Ang-2) and to uses of compounds having
such activities to treat diseases and disorders and find additional
compounds.
BACKGROUND OF THE INVENTION
[0003] Angiogenesis plays an important role in embryogenesis and
tumorigenesis. It is a complicated multistep process, which
includes the dynamic changes of cell-cell and cell-matrix
interactions, endothelial cell proliferation and migration,
recruitment of the peri-vascular supporting cells, and the
maturation process. Numerous molecules are involved in those
processes, including growth factors and their receptors, proteases,
adhesion receptors, and the ECM1 components. VEGF and angiopoietin
families play special roles in angiogenesis due to the restricted
expression of their receptors.
[0004] Ang-1 and Ang-2 are .about.70 kDa with a considerable
sequence homology, which consists of a signal peptide, an
N-terminal coiled-coil domain, a short linker peptide region, and a
C-terminal fibrinogen homology domain (FHD). The coiled-coil region
is responsible for dimerization/multimerization of angiopoietins,
and the fibrinogen homology domain binds to Tie-2 receptor. Both
Ang-1 and Ang-2 form dimers and oligomers.
[0005] Ang-1 and Ang-2 are the unique antagonists. Ang-1 induces
tyrosine phosphorylation of Tie-2 receptor and promotes recruitment
of the pericytes and smooth muscle cells, thereby playing a role in
establishing and maintaining the vascular integrity and quiescence.
As an antagonist of Ang-1, Ang-2 competes with Ang-1 for binding of
Tie-2, blocks the phosphorylation of Tie-2 receptors induced by
Ang-1, and loosens the interactions between endothelial and
peri-vascular support cells and ECM.
[0006] Targeted disruption of Ang-1 and Tie-2 and overexpression of
Ang-2 resulted in embryonic death with the similar vascular
defects. Those mice have normal primary vascular development, but
the remodeling and maturation of the vasculature are defective. The
transgenic mice overexpressing Ang-1 displayed increased
vascularization and decreased adult vasculature leakage. Together,
those results indicated that Ang-1 plays an indispensable role in
the formation of blood vessels during mouse development by
recruiting and maintaining peri-endothelial support cells.
[0007] Several studies have offered possible mechanisms for the
pro-angiogenic effect of Ang-1. Although Ang-1 does not stimulate
the proliferation of endothelial cells, it stimulates endothelial
cell migration, induces the capillary-like tubule formation, and
promotes survival of endothelial cells. Ang-1 inhibits apoptosis of
the endothelial cells via phosphatidylinositol 3-kinase/Akt
pathway.
[0008] Angiogenesis is regulated by the precise balance between
pro- and anti-angiogenic factors. Ang-2 expression is often induced
in the endothelia undergoing active remodeling or regression and by
hypoxia and several growth factors, including VEGF. Ang-2
destabilizes the vasculature. Thus, it initiates angiogenesis in
the presence of VEGF, which supplies endothelial cells with
necessary survival and proliferation signals, or induces apoptosis
of endothelial cells in the absence of the pro-angiogenic
factors.
SUMMARY OF THE INVENTION
[0009] One aspect of the present invention relates to
pharmaceutical compositions that comprise a pharmaceutically
acceptable carrier and either a therapeutically effective amount of
an ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1
and/or SEQ ID NO:2 or a homologous peptide thereof.
[0010] Another aspect of the present invention relates to
pharmaceutical compositions that comprise a pharmaceutically
acceptable carrier and a vector comprising a nucleic acid molecule
that comprises the nucleotide sequence that encodes an ECM-binding
fragment of Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID
NO:2 or a homologous peptide thereof.
[0011] A further aspect of the present invention provide methods of
treating an individual suspected of having coronary artery disease,
vascular disease or a condition involving ischemia. In some
embodiments, the methods comp se the step of administering to the
individual a pharmaceutical composition that comprises a
therapeutically effective amount of an ECM-binding fragment of
Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 or a
homologous peptide thereof. In some embodiments, the methods
comprise the step of administering to the individual pharmaceutical
compositions that comprises a vector comprising a nucleic acid
molecule that comprises the nucleotide sequence that encodes an
ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1
and/or SEQ ID NO:2 or a homologous peptide thereof.
[0012] Another aspect of the invention provides methods of
promoting angiogenesis, endothelial survival and maintaining
vascular integrity in an individual. In some embodiments, the
methods comprise the step of administering to the individual a
pharmaceutical composition that comprises a therapeutically
effective amount of an ECM-binding fragment of Ang-1 protein that
comprises SEQ ID NO:1 and/or SEQ ID NO:2 or a homologous peptide
thereof. In some embodiments, the methods comprise the step of
administering to the individual pharmaceutical compositions that
comprises a vector comprising a nucleic acid molecule that
comprises the nucleotide sequence that encodes an ECM-binding
fragment of Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID
NO:2 or a homologous peptide thereof.
[0013] According to some other aspects of the invention, methods
are provided to identify compounds that modulates binding of Ang-1
to ECM. The methods comprise performing a test assay that comprises
the steps of contacting a protein that comprises at least an
ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1
and/or SEQ ID NO:2 with ECM material in the presence of a test
compound, then measuring the level of binding of the protein that
comprises at least an ECM-binding fragment of Ang-1 protein that
comprises SEQ ID NO:1 and/or SEQ ID NO:2 with the ECM; and then
comparing the level with the level of binding of protein that
comprises at least an ECM-binding fragment of Ang-1 protein that
comprises SEQ ID NO:1 and/or SEQ ID NO:2 with ECM material in the
absence of the test compound. When the level of binding of the
protein that comprises at least an ECM-binding fragment of Ang-1
protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with the ECM
in the presence of the test compound is less than the level of
binding of the protein that comprises at least an ECM-binding
fragment of Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID
NO:2 with the ECM in the absence of the test compound results
indicate that the test compound modulates binding of Ang-1 to ECM
by inhibiting the binding. When the level of binding of the protein
that comprises at least an ECM-binding fragment of Ang-1 protein
that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with the ECM in the
presence of the test compound is more than the level of binding of
the protein that comprises at least an ECM-binding fragment of
Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with
the ECM in the absence of the test compound results indicate that
the test compound modulates binding of Ang-1 to ECM by enhancing
the binding.
[0014] A further aspect of the invention provides pharmaceutical
compositions which comprise a therapeutically effective amount of
Ang-2 protein and/or a vector comprising a nucleic acid molecule
that comprises the nucleotide coding sequence of Ang-2.
[0015] Additional aspects of the invention provide for methods of
treating an individual suspected of having cancer. The methods
comprise the step of administering to the individual a
pharmaceutical composition comprising a pharmaceutical composition
which comprise a therapeutically effective amount of Ang-2 protein
and/or a vector comprising a nucleic acid molecule that comprises
the nucleotide coding sequence of Ang-2.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIGS. 1Aa, 1Ab, 1B and 1C disclose data related to
angiopoietin-1 incorporated into the extracellular matrix. The
expression of Ang-1 and Ang-2 in the serum-fee conditioned media
(FIGS. 1Aa, 1Ab) and in the ECM (FIG. 1B) by the transfected LLC
and TA3 cells were determined by Western blot analyses using
anti-v5 antibody (Invitrogen). The Western blot analyses were
performed under non-reducing conditions using the concentrated
serum-free media (FIGS. 1Aa, 1Ab) or the ECM extracts (FIG. 1B)
derived from two independent isolates of the transfected LLC cells
expressing Ang-1v5 (FIG. 1Aa lanes 1 and 2, and FIG. 1B, lanes 1
and 2) or Ang-2v5 (FIG. 1Aa lanes 3 and 4, FIG. 1B lanes 3 ad 4);
two independent isolates of the transfected TA3 cells expressing
Ang-1v5 (FIG. 1Ab lanes 1 and 2, FIG. 1B lanes 6 and 7) or Ang-2v5
(FIG. 1Ab lanes 3 and 4, FIG. 1B lanes 8 and 9), and the tumor
cells transfected with the expression vector only (LLC, carcinoma
cells, FIG. 1Aa lane 5, FIG. 1B, lane 5; and TA3 cells, FIG. 1Ab
lane 5, FIG. 1B, lane 10). FIG. 1C, the same protein samples in the
FIG. 1B were subjected to -mercaptoethanol (5%) treatment.
Molecular mass markers are as indicated. kd indicates
kilodalton.
[0017] FIG. 2 shows data related to the ECM association of
angiopoietin-1. The Ang-1-containing ECM was extracted at room
temperature for 10 min with 0.15, 0.5, and 1 M NaCl and 0.5 and 1%
DOC, respectively. The insoluble ECM components were then extracted
with 1.times.SDS Laemmli buffer and subjected to Western blot
analysis using anti-v5 antibody (lanes 2-6, respectively). The
proteins in the lane 1 were derived from the ECM without prior
extraction with any reagents. Lanes 7 and 8 are the ECM extracts
derived from LLC cells expressing Ang-2 or transfected with the
expression vector alone, respectively. The molecular mass markers
are as indicated.
[0018] FIGS. 3A and 3B show data demonstrating Ang-1 does not bind
to heparin. In FIG. 3A, the purified v5-tagged Ang-1 proteins (500
ng/ml) were loaded onto a heparin-Sepharose CL-6B affinity column.
The flow-through was collected, and the column was washed with
0.15, 0.3, 0.5, and 1 M sodium chloride (NaCl, FIG. 3A, lanes 3-6),
and the eluted proteins were collected. All the collected fractions
were subjected to Western blot analysis using anti-v5 antibody. The
results indicated that Ang-1v5 does not bind to heparin-Sepharose,
and all the v5-tagged Ang-1 was in the flow-through fraction (FIG.
3A, lane 2). Lane 1 in FIG. 3A represents the starting materials.
In FIG. 3B, the ECM proteins were extracted, and Western blot
analysis was performed after the incubation of the confluent LLC
cells expressing Ang-1 with serum-free cell culture media
containing heparin (200 .mu.g/ml, FIG. 3B, lane 2), chondroitin
sulfate (200 .mu.g/ml, FIG. 3B, lane 3), or SFM alone (FIG. 3B,
lane 1) for 12 h. Molecular mass markers are as indicated.
[0019] FIG. 4 shows binding of Ang-1 to the different ECM
components. The binding affinities of Ang-1 to several different
ECM components were determined in the solid phase binding assays.
Ang-1 exhibited no affinity to fibronectin, laminin, collagen types
I and IV (columns 1-4, respectively), heparin, chondroitin sulfate,
and hyaluronic acid (columns 10 and 12, respectively). The weak
affinities of Ang-1 to vitronectin and fibrinogen were observed
(columns 5 and 6). Ang-1 displayed a moderate affinity to Matrigel
(column 7) and a strong affinity to the 2 M urea ECM extracts
derived from LLC carcinoma cells (column 8). All the experiments
were performed in triplicate.
[0020] FIGS. 5Aa, 5Ab, 5Ac, 5Ad, 5Ba, 5Bb, 5Bc, 5Bd and 5Be show
Ang-1 displayed a distinct ECM distribution pattern. The
distribution of Ang-1 in LLC cells expressing Ang-1v5 was
investigated by immunocytochemistry using anti-v5 antibody (FIG.
5Aa). The distribution was compared with that of fibronectin (FIG.
5Ba), laminin (FIG. 5Bb), collagen type I (FIG. 5Bc) and type IV
(FIG. 5Bd), and Ang-2 in LLC cells expressing Ang-2v5 (FIG. 5Ac).
The cell-free ECM derived from the LLC carcinoma cells expressing
Ang-1 (FIG. 5Ab) or Ang-2 (FIG. 5Ad) was also analyzed by
immunocytochemistry using anti-v5 antibody. LLC carcinoma cells
stained with the FITC-conjugated rabbit anti-mouse secondary
antibody only are shown in FIG. 5Be. Bar, 40 .mu.m.
[0021] FIG. 6 shows ECM-associated Ang-1 is released in the
response to PMA stimulation. Several factors were tested for their
abilities to release the ECM-sequestered Ang-1. After the
incubation of the LLC carcinoma cells expressing Ang-1 with SFM
alone (lane 1) or SFM containing PMA (0.5 .mu.g/ml, lane 2) TGF-1
(0.5 ng/ml, lane 3), bFGF (1 ng/ml, lane 4), epidermal growth
factor (10 ng/ml, lane 5), heparin-binding epidermal growth factor
(10 ng/ml, lane 6), and TGF-2 (0.5 ng/ml, lane 7), respectively,
for 14 h, the cells were released from the cell culture dishes by
the treatment of EDTA, and the remaining ECM components were
extracted with 1.times.SDS Laemmli buffer and subjected to the
Western blot analysis using anti-v5 antibody. Molecular mass
markers are as indicated.
[0022] FIGS. 7A, 7Ba, 7Bb, 7Bc and 7Bd shows ECM-associated Ang-1
exhibited no affinity to Tie-2-Fc fusion protein. A, Tie-2-Fc
fusion protein is capable of precipitating Ang-1 proteins from the
protein extracts, which were derived from the 2 M urea extraction
of the ECM of LLC carcinoma cells expressing Ang-1 (A, lane 2). The
starting materials for the immunoprecipitation were loaded in lane
1 (A), and the control for the immunoprecipitation contains
everything except Tie-2-Fc fusion proteins (A, lane 3). Molecular
mass markers are as indicated. B, the LLC cells expressing Ang-1 or
the ECM derived from LLC carcinoma cells expressing Ang-1 were
incubated with Tie-2-Fc fusion proteins (B, c and d) or anti-v5
antibody (B, a and b), and FITC-conjugated rabbit anti-human Fc or
anti-mouse secondary antibodies were used, respectively. Bar, 30
.mu.m.
[0023] FIGS. 8A and 8B show Tie-2 receptors on HUVECs
phosphorylated upon the adherence of HUVECs to the Ang-1-containing
ECM. The serum-starved HUVECs were lifted by the treatment of
EDTA/Hanks' balanced salt solution, and 1.times.106 of the HUVECs
were seeded into the plastic dish containing SFM with (200 ng/ml,
lane 3) or without soluble Ang-1 (lane 1) or the dishes coated with
the ECM derived from LLC cells expressing Ang-2 (lane 2) or Ang-1
in the presence (lane 5, 2 .mu.g/ml) or absence of Tie-2-Fc fusion
proteins (lane 4) for 30 min. The HUVECs were lysed, and 50 .mu.g
of proteins from each lysate were subjected to Western blot
analysis using anti-Tie-2 antibody (Santa Cruz Biotechnology, FIG.
8B). The rest of the proteins were used in the immunoprecipitation
using anti-Tie-2 antibody (Santa Cruz Biotechnology). The
immunoprecipitated proteins were subjected to Western blot analysis
using anti-phosphotyrosine (FIG. 8A). Molecular mass markers are as
indicated.
[0024] FIGS. 9Aa, 9Ab, 9Ac, 9Ad, 9B and 9C show the linker peptide
region of Ang-1 is responsible for its ECM association. Several
cDNA expression constructs were made including the full-length of
Ang-1 or Ang-2, the coiled-coil region, the coiled-coil plus the
linker peptide region, the fibrinogen-like region of Ang-1 (FIGS.
9Aa, 9Ab, 9Ac, 9Ad). All the Ang-1 fragments contain N-terminal
signal peptides of Ang-1 and the C-terminal v5 epitope tags. These
expression constructs were used to transfect COS-7 cells
transiently. 72 h after the transfection, the cell culture
supernatants (FIG. 9B) and the ECM proteins (FIG. 9C) were
collected or extracted and subjected to Western blot analyses to
determine the distribution of Ang-1, Ang-2, and the Ang-1 fragments
using anti-v5 antibody. Lanes 1 and 2, full-length Ang-1; lanes 3
and 4, full-length Ang-2; lanes 5 and 6, the coiled-coil domain
plus the linker peptide region of Ang-1; lanes 7 ad 8, the coiled
and coil region of Ang-1; lanes 9 and 10, the fibrinogen homology
domain of Ang-1; lane 11, COS-7 cells transfected with the
expression vector alone. Molecular mass markers are as
indicated.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0025] As used herein, the term "ECM" refers to the extracellular
matrix. The extracellular matrix (ECM) is a complex structural
entity surrounding and supporting cells that are found within
mammalian tissues. The EOM is comprises, structural proteins
(collagen and elastin), specialized proteins (e.g. fibrillin,
fibronectin, and laminin), and proteoglycans. Proteoglycans are
composed of a protein core to which is attached long chains of
repeating disaccharide units termed of glycosaminoglycans (GAGs)
forming extremely complex high molecular weight components of the
ECM.
[0026] The present invention relates to peptides and methods using
those peptides to treat an individual suspected of having cancer,
coronary artery disease, ischemias, and other vascular diseases.
Although the specific procedures and methods described herein are
exemplified using several specific peptides derived from
Angiopoietin-1 and -2, they are merely illustrative for the
practice of the invention. Analogous procedures and techniques, as
well as functionally equivalent peptides and peptide homologues, as
will be apparent to those of skill in the art based on the detailed
disclosure provided herein are also encompassed by the
invention.
[0027] Aspects of the present invention arises from the discovery
that angiopoietin-1 (Ang-1) associates with extracellular matrix
(ECM), and the function of Ang-1 is regulated by the association
with the ECM. Thus, while Ang-1 promotes angiogenesis when not
associated with the ECM, it is inhibited from promoting
angiogenesis while associated with the ECM.
[0028] According to some embodiments the present invention provides
for pharmaceutical compositions comprising a pharmaceutically
acceptable carrier and a therapeutically effective amount of an
ECM-binding fragment of the Ang-1 protein and/or a vector
comprising a nucleic acid molecule that comprises the nucleotide
sequence that encodes an ECM-binding fragment of Ang-1 protein.
[0029] As used herein, the term "ECM-binding fragment of Ang-1
protein" refers to any peptide sequence that comprises a peptide
fragment from Ang-1 that can bind to the ECM. In some embodiments
the ECM-binding fragment of Ang-1 protein is the 26-mer peptide
VHNLVSLCTKEGVLLKGGKREEEKPF (SEQ ID NO:1). In some embodiments the
ECM binding fragment of Ang-1 protein is VHNLVNLCTKEGVLLKGGKREEEKPF
(SEQ ID NO:2). The fragment may be the entire Ang-1 protein or it
may be a fragment of the Ang-1 protein. In some embodiments, the
ECM-binding fragment of Ang-1 protein may be a part of a fusion
protein that comprises Ang-1 protein sequence and non-Ang-1 protein
sequence. In some embodiments the ECM-binding fragment of Ang-1
protein is at least 26, at least 50, at least 75, at least 100, at
least 500, at least 1000 amino acid residues long. In some
embodiments the ECM-binding fragment of Ang-1 protein comprises SEQ
ID NO:4, SEQ ID NO:6, and/or SEQ ID NO:8
[0030] As used herein, the term "homologous peptide" refers to a
peptide that has at least 50% similarity to the peptide being
referred to. In some embodiments the peptide has at least 60%, at
least 70%, at least 80%, at least 90%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99%, or at least 100%
similarity to the ECM-binding fragment of Ang-1 protein and can
bind to the ECM. In some embodiments the homologous peptide has at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or at least
100% similarity to SEQ ID NO:1, SEQ ID NOs:2, SEQ ID NO:4, SEQ ID
NO:6, and/or SEQ ID NO:8. The homologous peptide may be isolated or
incorporated into another protein so that a fusion protein is
created.
[0031] According to some embodiments a homologous peptide refers to
a peptide that has conservative substitutions. A conservative
substitution is recognized in the art as a substitution of one
amino acid for another amino acid that has similar properties. In
some embodiments examples of conservative substitutions are those
that are described in Table I.
TABLE-US-00001 TABLE I Amino Acid Conservative Changes Alanine (A)
Glycine (G), Serine (S) Aspartic Acid (D) Glutamic Acid (E)
Glutamic Acid (E) Aspartic Acid (D) Phenylalanine (F) Tryptophan
(W), Tyrosine (Y) Glycine (G) Alanine (A) Histidine (H) Tyrosine
(Y) Isoleucine (I) Leucine (L), Methionine (M), Valine (V) Lysine
(K) Arginine (R) Leucine (L) Isoleucine (I), Methionine (M) Valine
(V) Methionine (M) Isoleucine (I), Leucine (L), Valine (V)
Asparagine (N) Glutamine (Q) Glutamine (Q) Asparagine (N) Arginine
(R) Lysine (K) Serine (S) Alanine (A), Threonine (T) Threonine (T)
Serine (S) Valine (V) Isoleucine (I), Methionine (M) Valine (V)
Tryptophan (W) Phenylalanine (F), Tyrosine (Y) Tyrosine (Y)
Phenylalanine (F) Histidine (H) Tryptophan (W)
[0032] As used herein, the phrase "homologous", "homologous
peptide", "homologous peptide thereof" or variations thereof,
refers to sequences characterized by a homology, at the nucleotide
level or amino acid level, of at least a specified percentage.
Homologous nucleotide sequences include those sequences coding for
isoforms of proteins. Such isoforms can be expressed in different
tissues of the same organism as a result of, for example,
alternative splicing of RNA. Alternatively, isoforms can be encoded
by different genes. Homologous nucleotide sequences include
nucleotide sequences encoding for a protein of a species other than
humans, including, but not limited to, mammals. Homologous
nucleotide sequences also include, but are not limited to,
naturally occurring allelic variations and mutations of the
nucleotide sequences set forth herein. Homologous amino acid
sequences include those amino acid sequences which contain
conservative amino acid substitutions and which polypeptides have
the same binding and/or activity.
[0033] Percent homology, similarity, or identity can be determined
by, for example, the Gap program (Wisconsin Sequence Analysis
Package, Version for Unix, Genetics Computer Group, University
Research Park, Madison Wis.), using default settings, which uses
the algorithm of Smith ad Waterman (Adv. Appl. Math., 1981, 2,
482-489). In some preferred embodiments, homology between the probe
and target is between about 50% to about 60%. In some embodiments,
nucleic acids have nucleotides that are about 60%, preferably about
70%, more preferably about 80%, more preferably about 85%, more
preferably about 90%, more preferably about 92%, more preferably
about 94%, more preferably about 95%, more preferably about 97%,
more preferably about 98%, more preferably about 99% ad most
preferably about 100% homologous to nucleotide sequences disclosed
herein.
[0034] Homology may also be at the polypeptide level. In some
embodiments, polypeptides are about 50%, about 60%, preferably
about 70%, more preferably about 80%, more preferably about 85%,
more preferably about 90%, more preferably about 92%, more
preferably about 94%, more preferably about 95%, more preferably
about 97%, more preferably about 98%, more preferably about 99% and
most preferably about 100% homologous to the polypeptide sequences
disclosed herein.
[0035] As used herein, the term "fusion protein" refers to a
protein that comprises amino acids that are from at least two
different proteins. As an example, a fusion protein may comprise
the ECM-binding fragment of Ang-1 protein and kinase domain of
another protein. This example would be considered a fusion protein.
The fusion of two protein sequences can be in any orientation. The
ECM-binding fragment may be placed at the N-terminus of a fusion
protein or at the C-terminus of a fusion protein. In some
embodiments, the ECM-binding fragment of Ang-1 protein may be
placed in the middle of a protein.
[0036] As used herein, the term "homologous peptide thereof" refers
to a peptide that is a homologous peptide, as defined above, to the
ECM-binding fragment of Ang-1 protein. The homologous peptide
thereof may be fusion protein, the entire Ang-1 protein, or a
fragment thereof.
[0037] As used herein, the term "nucleotide sequence that encodes
an ECM-binding fragment of Ang-1 protein" refers to a nucleotide
sequence that when transcribed and translated would comprise an
ECM-blinding fragment of Ang-1 protein. According to some
embodiments of the present invention, the nucleotide sequence may
be the entire sequence of Ang-1. In some embodiments the nucleotide
sequence may comprise a fragment of the nucleotide sequence of
Ang-1. In some embodiments the nucleotide sequence that encodes an
ECM-binding fragment of Ang-1 protein comprises SEQ ID NOs: 3, 5,
and/or 7. The nucleotide sequence of Ang-1 is well known to one of
ordinary skill in the art.
[0038] As used herein, the term "pharmaceutical composition" refers
to compositions according to the invention including delivery
components in combination with nucleic acid molecules and/or
peptide molecules which further comprise a pharmaceutically
acceptable carriers or vehicles, such as, for example, saline. Any
medium may be used which allows for successful delivery of the
peptide and/or nucleic acid. One skilled in the art would readily
comprehend the multitude of pharmaceutically acceptable media that
may be used in the present invention.
[0039] Pharmaceutical compositions may be formulated by one having
ordinary skill in the art with compositions selected depending upon
the chosen mode of administration. Suitable pharmaceutical carriers
are described in Remington's Pharmaceutical Sciences, A. Osol, a
standard reference text in this field, which is incorporated herein
by reference.
[0040] The pharmaceutical compositions of the present invention may
be administered by any means that enables the active agent to reach
the agent's site of action in the body of an individual.
Pharmaceutical compositions may be administered parenterally, i.e.,
intratumor, intravenous, subcutaneous, intramuscular. Intravenous
and intratumor administration are preferred routes. Dosage varies
depending upon known factors such as the pharmacodynamic
characteristics of the particular agent, and its mode and route of
administration; age, health, and weight of the recipient; nature
and extent of symptoms, kind of concurrent treatment, frequency of
treatment, and the effect desired.
[0041] As used herein, the term "vector" refers to a delivery
vehicle that is capable of delivering a nucleic acid to a cell. In
some embodiments, the vector is a viral vector. In general, viral
vectors may be DNA viruses such as recombinant adenoviruses and
recombinant vaccinia viruses or RNA viruses such as recombinant
retroviruses. Other recombinant vectors include recombinant
prokaryotes that can infect cells and express recombinant genes. In
addition to recombinant vectors, other vectors are also
contemplated such as encapsulation in liposomes,
lipofectin-mediated transfection, transferrin-mediated transfection
and other receptor-mediated means. In some embodiments the vector
is a DNA plasmid. The invention is intended to include such other
forms of expression vectors and other suitable delivery means which
serve equivalent functions and which become known in the at
subsequently hereto.
[0042] Examples of recombinant adenoviral vectors include those
which have the E1a region deleted and which carry a
temperature-sensitive mutation in E2a (Engelhardt et al., Hum Gene
Ther 5:1217-1229, 1994, which is incorporated herein by reference).
Other examples of recombinant adenoviral vectors useful to deliver
nucleic acid sequence of the present invention are described in
U.S. Pat. Nos. 5,756,283 and 5,707,618, which are each incorporated
herein by reference.
[0043] In another preferred embodiment of the present invention,
RNA is delivered to competent host cells by means of a retrovirus.
One skilled in the art would readily understand this technique of
delivering RNA to a host cell by such means. Any retrovirus which
serves to express the protein encoded by the RNA is intended to be
included in the present invention.
[0044] In another preferred embodiment of the present invention,
nucleic acid is delivered through folate receptor means. The
nucleic acid sequence to be delivered to a host cell is linked to
polylysine and the complex is delivered to the tumor cell by means
of the folate receptor. U.S. Pat. No. 5,108,921 issued Apr. 28,
1992 to Low et al., which is incorporated herein by reference,
describes such delivery components.
[0045] In another preferred embodiment of the present invention,
nucleic acid is delivered through the use of lipofectin-mediated
DNA transfer. LipofectAMINE.TM. liposome reagent (Life
Technologies, Gaithersburg Md.) is a commercially available
liposome encapsulation reagent which can be used for encapsulating
cells following manufacturer's instructions. LipofectAMINE.TM.
liposome reagent encapsulated nucleic acid molecules may be
delivered to a host cell using liposome formulation administration
methods.
[0046] In another preferred embodiment of the present invention,
nucleic acid is delivered through the use of cationic
lipid-mediated DNA transfer such as that which is described in U.S.
Pat. No. 5,703,055, which is incorporated herein by reference.
[0047] In another preferred embodiment of the present invention,
nucleic acid is delivered through the use of liposome-mediated DNA
transfer such as that which is described in U.S. Pat. Nos.
4,235,871, 4,241,046 and 4,394,448, which are each incorporated
herein by reference.
[0048] According to some embodiments the present invention provides
methods of treating an individual suspected of having coronary
artery disease, vascular disease, or a condition involving
ischemia. In some embodiments the method comprises the steps of
administering to the individual a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and a
therapeutically effective amount of an ECM-binding fragment of
Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 or a
homologous peptide thereof and/or a vector comprising a nucleic
acid molecule that comprises the nucleotide sequence that encodes
an ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1
and/or SEQ ID NO:2 or a homologous peptide thereof. In some
embodiments, the methods are provided for individuals need
thereof.
[0049] As used herein, the term "coronary artery disease" refers to
diseases that are a result of the buildup of cholesterol in the
inside layers of the arteries. As used herein, the term "vascular
disease" refers to diseases that is related to the circulatory
system. As used herein, the term "ischemia" refers to a condition
that is a caused by a lack of blood flow that would otherwise be
present in a healthy individual.
[0050] In some embodiments the compounds of the invention, may be
administered to a subject per se or in the form of a pharmaceutical
composition. Pharmaceutical compositions comprising the compounds
of the invention may be manufactured by means of conventional
mixing, dissolving, granulating, dragee-making, levigating,
emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions may be formulated in conventional
manner using one or more physiologically acceptable carriers,
diluents, excipients or auxiliaries which facilitate processing of
the active peptides or peptide analogues into preparations which
can be used pharmaceutically. Proper formulation is dependent upon
the route of administration chosen.
[0051] For topical administration the compounds of the invention
may be formulated as solutions, gels, ointments, creams,
suspensions, etc. as are well-known in the art.
[0052] Systemic formulations include those designed for
administration by injection, e.g. subcutaneous, intravenous,
intramuscular, intrathecal or intraperitoneal injection, as well as
those designed for transdermal, transmucosal, oral or pulmonary
administration.
[0053] For injection, the compounds of the invention may be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. The solution may contain formulatory
agents such as suspending, stabilizing and/or dispersing
agents.
[0054] Alternatively, the compounds may be in powder form for
constitution with a suitable vehicle, e.g., sterile pyrogen-free
water, before use.
[0055] For transmucosal administration, penetrants appropriate to
the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art.
[0056] For oral administration, the compounds can be readily
formulated by combining the active peptides or peptide analogues
with pharmaceutically acceptable carriers well known in the art.
Such carriers enable the compounds of the invention to be
formulated as tablets, pills, dragees, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a
patient to be treated. For oral solid formulations such as, for
example, powders, capsules and tablets, suitable excipients include
fillers such as sugars, such as lactose, sucrose, mannitol and
sorbitol; cellulose preparations such as maize starch, wheat
starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP);
granulating agents; and binding agents. If desired, disintegrating
agents may be added, such as the cross-linked polyvinylpyrrolidone,
agar, or alginic acid or a salt thereof such as sodium
alginate.
[0057] If desired, solid dosage forms may be sugar-coated or
enteric-coated using standard techniques.
[0058] For oral liquid preparations such as, for example,
suspensions, elixirs and solutions, suitable carriers, excipients
or diluents include water, glycols, oils, alcohols, etc.
Additionally, flavoring agents, preservatives, coloring agents and
the like may be added.
[0059] For buccal administration, the compounds may take the form
of tablets, lozenges, etc. formulated in conventional manner.
[0060] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray from pressurized packs or a nebulizer,
with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0061] The compounds may also be formulated in rectal or vaginal
compositions such as suppositories or retention enemas, e.g,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0062] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0063] Alternatively, other pharmaceutical delivery systems may be
employed. Liposomes and emulsions are well known examples of
delivery vehicles that may be used to deliver peptides and/or
nucleotides of the invention. Certain organic solvents such as
dimethylsulfoxide also may be employed, although usually at the
cost of greater toxicity. Additionally, the compounds may be
delivered using a sustained-release system, such as semipermeable
matrices of solid polymers containing the therapeutic agent.
Various of sustained-release materials have been established and
are well known by those skilled in the art. Sustained-release
capsules may, depending on their chemical nature, release the
compounds for a few weeks up to over 100 days. Depending on the
chemical nature and the biological stability of the therapeutic
reagent, additional strategies for protein stabilization may be
employed.
[0064] According to some embodiments of the present invention the
pharmaceutical composition is administered in an amount that is
therapeutically effective. As used herein, the term
"therapeutically effective" refers to an amount effective to
achieve the intended purpose. In some embodiments the intended
purpose is to treat coronary artery disease, vascular disease, a
condition involving ischemia, or cancer. In some embodiments a
therapeutically effective amount refers to an amount effective to
ameliorate or prevent the symptoms, or prolong the survival of the
patient being treated. Therapeutically effective amounts are
typically determined by the effect they have compared to the effect
observed when a composition which includes no active ingredient is
administered to a similarly situated individual The precise
effective amount for a subject will depend upon the subject's size
and health, the nature and extent of the condition, and the
therapeutics or combination of therapeutics selected for
administration. However, the effective amount for a given situation
is determined by routine experimentation and is within the judgment
of the clinician. Determination of a therapeutically effective
amount is well within the capabilities of those skilled in the art,
especially in light of the detailed disclosure provided herein.
[0065] Initial dosages can also be estimated from in vivo data,
e.g., animal models, using techniques that are well known in the
art. One having ordinary skill in the art could readily optimize
administration to humans based on animal data.
[0066] Dosage amount and interval may be adjusted individually to
provide plasma levels of the compounds which are sufficient to
maintain therapeutic effect. Usual patient dosages for
administration by injection range from about 0.1 to 5 mg/kg/day,
preferably from about 0.5 to 1 mg/kg/day. Therapeutically effective
serum levels may be achieved by administering multiple doses each
day.
[0067] In cases of local administration or selective uptake, the
effective local concentration of the compounds may not be related
to plasma concentration. One having skill in the art will be able
to optimize therapeutically effective local dosages without undue
experimentation.
[0068] The amount of compound administered will, of course, be
dependent on the subject being treated, on the subject's weight,
the severity of the affliction, the manner of administration and
the judgment of the prescribing physician.
[0069] The therapy may be repeated intermittently while symptoms
detectable or even when they are not detectable. The therapy may be
provided alone or in combination with other drugs.
[0070] According to some embodiments, the present invention
provides for methods of promoting angiogenesis, endothelial
survival, and/or maintaining vascular integrity comprising the
administration of a pharmaceutical composition comprising a
pharmaceutically acceptable carrier and a therapeutically effective
amount of an ECM-binding fragment of Ang-1 protein that comprises
SEQ ID NO:1 and/or SEQ ID NO:2 or a homologous peptide thereof
and/or a vector comprising a nucleic acid molecule that comprises
the nucleotide sequence that encodes an ECM-binding fragment of
Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 or a
homologous peptide thereof.
[0071] As used herein, the term "angiogenesis" refers to the growth
of blood vessels. In some embodiments the promotion of angiogenesis
promotes the growth of new blood vessels, while in some embodiments
existing blood vessels are promoted to grow. Angiogenesis is a term
well understood by those of ordinary skill in the art. In some
embodiments endothelial survival refers to the process of
preventing endothelial cells from dying. In some embodiments
endothelial survival refers to the promoting the growth of
endothelial cells.
[0072] In some embodiments "maintaining vascular integrity" refers
to the process by which the a vascular system viability and
functions are kept at specific level. In some embodiments the
vascular system may be located throughout the individual. In some
embodiments the vascular system may be localized to a specific
region of the individual. For example, if a person has a poor
vascular system in the foot, the pharmaceutical composition may be
administered in a therapeutically effective amount to promote and
maintain vascular integrity in that foot, while the rest of the
vascular system may be unaffected. However, in other embodiments a
therapeutically effective amount may promote angiogenesis,
endothelial survival, and maintaining vascular integrity throughout
the individual.
[0073] According to some embodiments, the present invention
provides for methods for identifying compounds that modulates the
binding of Ang-1 to ECM comprising performing a test assay that
comprises the steps of contacting a protein that comprises at least
an ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1
and/or SEQ ID NO:2 with ECM material in the presence of a test
compound and measuring the level of binding of the protein that
comprises at least an ECM binding fragment of Ang-1 protein that
comprises SEQ ID NO:1 and/or SEQ ID NO:2 with the ECM. In some
embodiments the method further comprises comparing the level with
the level of binding of the protein that comprises at least an
ECM-binding fragment of Ang-1 protein that comprises SEQ ID NO:1
and/or SEQ ID NO:2 with ECM material in the absence of said test
compound. In some embodiments when the level of binding of the
protein that comprises at least an ECM-binding fragment of Ang-1
protein that comprises SEQ ID NO:1 and SEQ ID NO:2 with the ECM in
the presence of the test compound is less than the level of binding
of the protein that comprises at least an ECM-binding fragment of
Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO: 2 with
the ECM in the absence of the test compound results indicate that
the test compound modulates binding of Ang-1 to ECM by inhibiting
the binding.
[0074] In some embodiments when the level of binding of the protein
that comprises at least an ECM-binding fragment of Ang-1 protein
that comprises SEQ ID NO:1 and/or SEQ ID NO: 2 with the ECM in the
presence of the test compound is more than the level of binding of
the protein that comprises at least an ECM-binding fragment of
Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO: 2 with
the ECM in the absence of the test compound results indicate that
the test compound modulates binding of Ang-1 to ECM by enhancing
the binding.
[0075] As used herein, the term "modulates" refers to an increase
or a decrease. In some embodiments the test compound increases the
level of Ang-1 protein binding to the ECM. In some embodiments the
test compound decreases the level of Ang-1 protein binding to the
ECM.
[0076] As used herein, the term "at least an ECM-binding fragment
of Ang-1 protein" refers to a protein that comprises a fragment of
Ang-1 that can bind to ECM. In some embodiments this refers to SEQ
ID NO:1 and/or SEQ ID NO:2. In other embodiments, this refers to
protein that comprises a section of the protein that is homologous
to an ECM-binding fragment of Ang-1 protein. In some embodiments,
the protein can be the full-length Ang-1 protein or a fragment
thereof. In some embodiments the "at least an ECM-binding fragment
of Ang-1 protein" comprises SEQ ID NO:4, SEQ ID NO:6, and/or SEQ ID
NO:8. In some embodiments, the protein can be a fusion protein that
comprises Ang-1 protein sequence and non-Ang-1 protein sequence. In
some embodiments, a protein comprising at least an ECM-binding
fragment of Ang-1 protein comprises a peptide sequence that has at
least 50%, at least 60%, at least 70%, at least 80%, at least 90%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, at least 100% similarity to an ECM-binding fragment of Ang-1.
In some embodiments a protein comprising at least an ECM-binding
fragment of Ang-1 protein comprises a peptide sequence that has at
least 50%, at least 60%, at least 70%, at least 80%, at least 90%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or at least 100% similarity to SEQ ID NO:4, SEQ ID NO:6,
and/or SEQ ID NO:8. In some embodiments a protein comprising at
least an ECM-binding fragment of Ang-1 protein comprises a peptide
sequence that has at least 50%, at least 60%, at least 70%, at
least 80%, at least 90%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99%, or at least 100% similarity to SEQ ID
NO:1 and/or SEQ ID NO:2.
[0077] According to some embodiments "ECM material" refers to a
compound or material that is found in the extracellular matrix that
can bind to an ECM-binding fragment of Ang-1 protein. According to
some embodiments the "ECM material" refers to a composition
comprising fibronection, laminin, type I collagen, type IV
collagen, vitronectin, fibrinogen, matrigel, LLC carcinoma ECM,
BSA, heparin, chondroitin sulfate, or hyaluronic acid.
[0078] In some embodiments the ECM material is produced by
culturing cells on a substrate for a sufficient time for the cells
to produce the ECM material on the substrate and then removing the
cells from the substrate without removing the ECM material. In some
embodiments the cells that are used to produce the ECM material are
Lewis Lung carcinoma cells or TA3 murine mammary carcinoma cells.
As used herein, the term "substrate" refers to any vessel or
container that is capable of culturing cells. Examples of
substrates include, but are not limited to, petri dishes, 6-well
plates, 96-well plates, 384-well plates, and the like. Removing
cells from the substrate without removing the ECM material is well
within the skill of one of ordinary skill in the art. An example of
how to remove the cells without removing the ECM includes
contacting the cells with a chelator such as EDTA or EGTA for a
sufficient time to remove the cells without effecting the ECM
material. There are other methods that can performed the same
function as contacting the cells with EDTA or EGTA and are within
the scope of the current invention.
[0079] According to some embodiments the protein that comprises at
least an ECM-binding fragment of Ang-1 comprising a detectable
label. As used herein, the term "detectable label" refers to any
molecule that can be detected with methods that are well known to
those of ordinary skill in the art. Molecules with detectable
labels include without limitation proteins, protein fragments,
antibodies, fluorescent labels, radioactive labels, chromophores,
chemiluminescent probes, and the like. In some embodiments the
detectable label is used to measure the level of binding of the
protein that comprises at least an ECM-binding fragment of Ang-1
protein on the ECM.
[0080] In some embodiments the method of identifying compound that
modulates the binding of Ang-1 to ECM further comprises multiple
test assays that are identical except that the amount of the test
compound used differs. To aid in determining the effective amount
of a test agent multiple assays are preformed using different
amounts of the test compound. In some embodiments at least 2 assays
are performed. In some other embodiments at least 3, at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least
10, at least 15, at least 20, at least 30, at least 40, at least
50, at least 100 assays are performed using different amounts of
the test compound.
[0081] According to some embodiments the methods of identifying a
compound that modulates the binding of Ang-1 to ECM further
comprises determining the level of binding of a protein that
comprises at least an ECM-binding fragment of Ang-1 protein that
comprises SEQ ID NO:1 and/or SEQ ID NO:2 with ECM material in the
absence of the test compound by performing a control assay wherein
the control assay comprises the steps of contacting a protein that
comprises at least an ECM-binding fragment of Ang-1 protein that
comprises SEQ ID NO:1 and/or SEQ ID NO:2 with ECM material in the
absence of a test compound and measuring the level of binding of
the protein that comprises at least an ECM binding fragment of
Ang-1 protein that comprises SEQ ID NO:1 and/or SEQ ID NO:2 with
the ECM.
[0082] Assays that can be used for the methods to identify
compounds that modulates the binding of Ang-1 to ECM are well known
to those of ordinary skill in the art and require only routine
experimentation Examples of assays that are well known to those of
ordinary skill in the art include ELISA, Sandwich Assays, flow
cytometry, immunoprecipitation, and the like.
[0083] According to some embodiments the present invention provides
for pharmaceutical compositions comprising a pharmaceutically
acceptable carrier and a therapeutically effective amount of Ang-2
protein and/or a vector comprising a nucleic acid molecule that
comprises the nucleotide coding sequence of Ang-2.
[0084] As used herein, the term "Ang-2" refers to the protein or
nucleic acid encoding the protein or fragment thereof of
Angiopoietin-2. In some embodiments the Ang-2 is mammalian Ang-2.
In some embodiments, the Ang-2 is human, mouse, rat, dog, cat, pig,
or horse. In some embodiments the Ang-2 protein comprises SEQ ID
NO:10 and/or SEQ ID NO:12. In some embodiments the Ang-2 nucleotide
coding sequence comprises SEQ ID NO 9 and/or SEQ ID NO:11. In some
embodiments, the Ang-2 protein or the nucleic acid that encodes
Ang-2 is a fragment of the Ang-2 protein or the nucleotide coding
sequence of Ang-2. In some embodiments the Ang-2 protein comprises
a fragment of SEQ ID NO:10 and/or SEQ ID NO:12. In some
embodiments, "Ang-2" refers to a fusion protein comprising
non-Ang-2 protein sequence and Ang-2 protein sequence. According to
some embodiments, "Ang-2" refers to a protein that has at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
at least 100% similarity to SEQ ID NO:10 and/or SEQ ID NO:12. In
some embodiments the nucleotide coding sequence comprises a
nucleotide coding sequence that is at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99%, at least 100% identical
to SEQ ID NO:9 and/or SEQ ID NO:11.
[0085] According to some embodiments the present invention provides
for methods of treating an individual, or an individual in need
thereof, suspected of having cancer comprising the step of
administering to the individual a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and a
therapeutically effective amount of Ang-2 protein and/or a vector
comprising a nucleic acid molecule that comprises the nucleotide
coding sequence of Ang-2. The methods of administration are defined
above as well as what is meant by a therapeutically effective
amount.
[0086] The cancers that can be treated are not limited to any
cancer described herein and can include cancers of the bladder,
cancers of the brain, cancers of the breast, cancers of the colon,
hodgkin's disease, cancers of the kidney, cancers of the lung,
melanoma, non-hodgkin's lymphoma, oral cancer, ovarian cancer,
prostate cancer, uterine/cervical cancer, leukemia, cancers of the
pancreas, testicular cancer, solid tumors, and the like.
Examples
[0087] Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) affect
angiogenesis differently during embryogenesis and tumorigenesis. In
an attempt to understand the molecular basis underlying the
distinct roles of those two homologous molecules, we investigated
the association of Ang-1 and Ang-2 with the extracellular matrix
(ECM). TA3 murine mammary carcinoma (TA3) and Lewis lung carcinoma
cells expressing v5 epitope-tagged Ang-1 and Ang-2 were used in our
studies. The results indicated that Ang-1 is secreted and
incorporated into the ECM of the tumor cells, whereas Ang-2 is not
associated with the ECM. The mutagenesis study indicated the domain
that is responsible for the ECM association of Ang-1 is the linker
peptide region between the coiled-coil and the fibrinogen-like
domains. A weak binding between the coiled-coil domain of Ang-1 and
the ECM was observed. Immunocytochemistry study revealed a distinct
ECM distribution pattern of Ang-1, which is quite different from
that of fibronectin, laminin, and collagen types I and IV. The
ECM-associated Ang-1 proteins are released, and Tie-2 receptors are
phosphorylated upon the adhesion of human umbilical vein
endothelial cells. Implications of the difference in the ECM
association of Ang-1 and Ang-2, which are related to the regulation
of angiopoietin activity and their roles in local versus distant
angiogenesis during tumor metastasis, are discussed.
[0088] Ang-1 and -2 are expressed by tumor cells. We found that
overexpression of exogenous Ang-2, but not Ang-1, inhibits growth
and metastasis of Lewis lung carcinoma (LLC) and TA3 murine mammary
carcinoma cells. The tumors overexpressing Ang-2 exhibited aberrant
and incomplete angiogenesis in vivo, which is characterized by
formation of the disorganized endothelial cell aggregates, the lack
of endothelial associated smooth muscle cells, and massive
apoptosis of the endothelial cells and surrounding tumor cells.
This result is consistent with the notion that Ang-2 inhibits the
Ang-1-dependent recruitment of smooth muscle cells.
[0089] To reveal the molecular basis underlying the different roles
of angiopoietins in tumor angiogenesis, we investigated the
relationship between angiopoietins and the ECM in the present
study. We found that, unlike Ang-2, Ang-1 is secreted and
incorporated into the ECM via its linker peptide region. The
association between Ang-1 and the ECM is strong, and the
distribution of Ang-1 in the ECM is unique and different from that
of fibronectin, laminin, and collagen type I and type IV. The
releasing or incorporation of Ang-1 from or into the ECM is
regulated by different factors. Tie-2 phosphorylation was detected
in HUVECs seeded onto the ECM containing Ang-1, which indicates a
regulatory role of the ECM association of Ang-1 in tumor
angiogenesis.
[0090] Ang-2 expression is regulated by hypoxia and growth factors.
Unlike Ang-2, little is known about the regulation of Ang-1
expression. The finding reported herein offers a possible
regulatory mechanism for the availability of Ang-1 proteins, that
is instead of regulating the production of Ang-1, it may be
regulated by its ECM association. The strong ECM binding of Ang-1
implies the effect of Ang-1 is limited to the local environment
where it is produced, whereas Ang-2 can diffuse to and affect
angiogenesis in the distant sites.
Experimental Procedures
[0091] Cell Culture and Reagents--Lewis lung carcinoma (LLC, ATCC),
TA3 mammary carcinoma (37) cells, and the tumor cell transfectants
were maintained as described (17). Anti-laminin, -fibronectin
(Sigma), -collagen types I and IV (Biodesign international),
-phosphotyrosine (PY20, Calbiochem), -Tie-2 (C-20, Santa Cruz
Biotechnology), -Ang-1 (C-19, Santa Cruz Biotechnology), and -v5
antibodies were used.
[0092] Construction of the Expression Vectors--Full-length Ang-1
and Ang-2 cDNAs were generated by RT-PCR as described (17, 40). The
coiled-coil domain and the coiled-coil plus the linker peptide
region were generated by RT-PCR using the following pairs of
primers. The forward primer for the coiled-coil domain is
5'-ACAATGACAGTTTTCCTTTCCTTT-3' and the reverse primer is
5'-TGTGTCCATGAGCTCCAGTTGTTG-3'. The coiled-coil plus the linker
peptide region was amplified using the same forward primer as that
of the coiled-coil domain and a reverse primer,
5'-AAATGGTTTCTCTTCTTCTCTTTT-3'. The stop codons were omitted from
the reverse primers so that the v5 and 6.times. histidine tags in
the pEF6/V5-His expression vector (Invitrogen) can be attached to
the C terminus of the Ang-1 fragments. To fuse the FHD of Ang-1 to
the signal peptide of Ang-1, we used the full-length Ang-1 in the
expression vector (17) as a template and the ExSite PCR-based
site-directed mutagenesis kit (Stratagene) together with a forward
primer derived from the beginning of the FHD,
5'-CGAGACTGTGCAGATGTATATCAA-3', and a reverse primer derived from
the end of the signal peptide, 5'-TCTTCTCCCTCCGTTTTCTGGATT-3'. The
extracellular domain of Tie-2 was obtained by RT-PCR using the
following pair of primers. The forward primer is
5'-AGTATGGACTCTTTAGCCGGCTTA-3' and the reverse primer is
5'-CATCTTTCCCCCTCCGAGGTCTGC-3'. The PCR products were inserted in
frame into 5'-end of the Fc fragments of human IgG in pEF6/V5-His
expression vectors to generate the Tie-2-Fc fusion construct (40).
The authenticity and orientation of the cDNA inserts were confirmed
by DNA sequencing.
[0093] Transfection--LLC and TA3 carcinoma cells were transfected
with the expression constructs containing Ang-1, Ang-2, or the
expression vector alone. The transfected cells expressing Ang-1 and
Ang-2 were identified as described (17). COS-7 cells were used in
the transient transfection of Ang-1 and -2, the coiled-coil domain,
the coiled-coil plus linker region, and the FHD of Ang-1 using
LipofectAMINE (Life Technologies, Inc.) as described (17).
[0094] Preparation of the Secreted and the ECM-Associated Proteins
and Western Blot Analysis--The cell culture supernatants of LLC and
TA3 transfectants or the transiently transfected COS-7 cells were
collected. The confluent cells layers were released from the
culture dishes by incubating with PBS containing 5 mM EDTA. The ECM
components remaining on the culture dishes were washed and
extracted with 1.times.SDS Laemmli sample buffer with or without
5%-mercaptoethanol.
[0095] To determine the affinity of the ECM association of Ang-1,
the confluent LLC cells expressing Ang-1 were lifted as described.
The remaining ECM components were extracted with 0.5 M PBS and 1 M
sodium chloride (NaCl) and 0.5 and 1% deoxycholate (DOC) at room
temperature for 10 min. The remaining insoluble materials were
solubilized by 1.times.SDS Laemmli sample buffer and subjected to
Western blot analysis.
[0096] Immunocytochemistry--LLC cells expressing Ang-1 or Ang-2 or
transfected with the expression vectors were cultured in 35-mm
dishes until subconfluence or confluence. They were fixed with
methanol at 20.degree. C. for 15 min. Antibodies against v5
epitope, fibronectin, laminin, type I or IV collagens were used to
detected Ang-1v5, Ang-2v5, fibronectin, laminin, types I or IV
collagen, respectively, in those fixed cells.
[0097] Extraction of the ECM Components and Affinity
Purification--The ECM materials derived from the cultured LLC cells
were extracted overnight at 4.degree. C. in 2 M urea and 0.05 M
Tris-HCl (pH 7.4). The soluble materials were dialyzed against PBS
and used to coat the enzyme-linked immunosorbent assay plates. The
ECM derived from the LLC transfectants expressing Ang-1 was
extracted in the same way. The ECM extracts containing Ang-1
proteins and the serum-free culture media containing secreted
Ang-2, both of which contain v5 and 6.times. histidine tags at
their C-terminal ends, were loaded onto Ni+ Probond affinity
columns (Invitrogen) and purified following the manufacturer's
instructions.
[0098] Purified Ang-1v5/His proteins were loaded onto a
heparin-Sepharose CL-6B column to test their affinity to heparin.
The flow-through fraction was collected, and the column was washed
with different concentrations of NaCl from 0.15 to 1 M. The eluted
fractions were collected and, along with the flow-through
fractions, were subjected to Western blot analysis.
[0099] Solid Phase Binding Assay--96-Well enzyme-linked
immunosorbent assay plates were coated overnight at 4.degree. C.
with different ECM components. The components were fibronectin,
laminin, type I and IV collagens, Matrigel (20 .mu.g/ml, Becton
Dickinson), vitronectin (10 .mu.g/ml, Sigma), fibrinogen (20
.mu.g/ml, Sigma), heparin, chondroitin sulfate, hyaluronic acid
(200 .mu.g/ml, Sigma), and the whole ECM extracts derived from LLC
cells (100 .mu.g/ml). The coated plates were washed and blocked
with 0.5% bovine serum albumin. The affinity-purified Ang-1v5/His
and Ang-2v5/His were added into the coated plates (100 ng/ml) for
overnight incubation at 4.degree. C. After extensive washing, the
bound Ang-1 and Ang-2 were detected. The assays were performed in
triplicate.
[0100] Tie-2 Tyrosine Phosphorylation Assay--LLC carcinoma cells
expressing Ang-1 or Ang-2 were cultured in 100-mm dishes until
confluence and were lifted from the dishes as described. The dishes
containing the ECM components deposited by the cultured cells were
used immediately.
[0101] HUVECs (ATCC) were cultured until subconfluence, and
switched into the serum-free medium overnight. The cells were then
lifted with 0.53 mM EDTA in Hanks' balanced buffer (Life
Technologies, Inc.) and washed. 1.times.106 HUVECs were seeded onto
a plastic dish or one of the ECM-coated dishes freshly generated as
described above. They were cultured at 37.degree. C. in serum-free
medium with or without soluble Ang-1 (200 ng/ml) or Tie-2-Fc fusion
proteins (2 .mu.g/ml) for 30 min. The cells were then lysed at
4.degree. C. with the lysis buffer (50 mM Tris-HCl (pH 7.4), 50 mM
NaCl, 1% Triton X-100, 2 mM EDTA, 2 mM sodium orthovanadate, 2 mM
sodium fluoride, 2 mM phenylmethylsulfonyl fluoride, 1 mM
leupeptin, 1 mM pepstatin A, and 10 .mu.g/ml aprotinin). Tie-2
proteins were immunoprecipitated using anti-Tie-2 polyclonal
antibody (Santa Cruz Biotechnology). The immunoprecipitated
proteins were subjected Western blot analysis using
anti-phosphotyrosine antibody (Y20, Calbiochem).
Results
[0102] Angiopoietin-1, but not Angiopoietin-2, Binds to the
Extracellular Matrix--In the process of studying the role of
angiopoietins in tumor angiogenesis, we experienced difficulty
obtaining the transfected tumor cells that secrete high levels of
Ang-1, whereas the transfectants that secrete high levels of Ang-2
were easily obtained. One possible reason for the phenomena is that
Ang-1 and Ang-2 may associate with the ECM differently. To
investigate that, we generated the stably transfected Lewis lung
carcinoma (LLC) and TA3 murine mammary carcinoma (TA3) cells
expressing v5 epitope-tagged Ang-1 or Ang-2 (FIG. 1, A, a and b).
Under non-reducing conditions both Ang-1 and Ang-2 tend to
aggregate with each other to form dimers and oligomers (FIGS. 1, A
and B). The patterns of the aggregation are distinct between Ang-1
and Ang-2. Ang-1 tends to form higher order oligomers (FIG. 1A),
whereas Ang-2 forms dimers, trimers, and oligomers. The molecular
weight of the monomer of Ang-1 and Ang-2 is .about.70 kDa. Because
of the C-terminal v5 and 6.times. histidine epitope tags, Ang-1v5
and Ang-2v5 migrate a little slower.
[0103] The aggregation of Ang-1 and Ang-2 is sensitive to the
reducing agents, such as -mercaptoethanol. After boiling the
protein samples in 1.times. Laemmli SDS sample buffer containing
5%-mercaptoethanol, the aggregated Ang-1 and Ang-2 are dissociated
into monomers (data not shown). It was noted that the amount of the
secreted Ang-1 is often lower than that of Ang-2 derived from LLC
and TA3 transfectants (FIGS. 1A, a and b). To examine the
possibility that the lack of Ang-1 secretion is due to
incorporation of Ang-1 into the ECM of the tumor cells, the
transfectants expressing Ang-1 and Ang-2 were grown until
confluence, and the cells were then detached from the culture
dishes by the EDTA treatment, which is a standard procedure to
release cultured cells and leave the ECM components behind on the
culture dishes (38). The remaining ECM components were then
extracted from the culture dishes by 1.times.SDS Laemmli buffer
with or without -mercaptoethanol. Western blot analyses were
performed using anti-v5 monoclonal antibody to detect v5-tagged
Ang-1 or Ang-2. The results indicated that Ang-1 is present in the
ECM fraction of the transfected tumor cells, whereas Ang-2 is
absent (FIG. 1B). The ECM-associated Ang-1 is highly aggregated to
form oligomers, and no monomer is detected (FIG. 1B, lanes 1 and 2,
and 6 and 7). The aggregated Ang-1 oligomers were dissociated into
the monomers by the treatment with -mercaptoethanol (FIG. 1C),
which indicates the role of the cysteine residues in the
aggregations. Thus, we have established that Ang-1, but not Ang-2,
is incorporated into the ECM of those carcinoma cells.
[0104] To confirm that the endogenously expressed Ang-1 is
associated with the ECM as the transfected Ang-1, the polyclonal
anti-Ang-1 antibody (C19, Santa Cruz Biotechnology) was first
purified through an Ang-1v5-conjugated protein A affinity column.
The purified antibody was used in Western blot analysis of the ECM
components derived from HUVECs.
[0105] The results indicated that Ang-1 produced by HUVECs is
incorporated into the ECM (data not shown). Due to the superior
quality of the anti-v5 monoclonal antibody compared with that of
the polyclonal anti-Ang-1 antibody, even after the affinity
purification (Santa Cruz Biotechnology), most of the following
experiments were performed using the transfected tumor cells
expressing v5-tagged Ang-1 and Ang-2.
[0106] The Biochemical Characters of the ECM-associated Ang-1--The
strength of the ECM association of Ang-1 was tested by extracting
the cell-free ECM deposited on the culture dishes by LLC cells
expressing Ang-1v5 with different concentrations of sodium chloride
(NaCl) and deoxycholate (DOC). The insoluble materials after the
extractions were solubilized with 1.times.SDS Laemmli buffer and
subjected to Western blot analysis. As shown in FIG. 2, most of the
ECM-incorporated Ang-1 resisted 0.15 M NaCl extraction (FIG. 2,
lane 2); a fair amount of Ang-1 remains in the ECM after 1 M NaCl
extraction (FIG. 2, lane 4), and a fraction of Ang-1 is in the ECM
fraction even after 1% DOC extraction (FIG. 2, lane 6), which
indicated a strong ECM association and implicated a gradual
assembly process of Ang-1 into the ECM (FIG. 2).
[0107] The Binding Affinity of Ang-1 to Different ECM
Components--The C terminus of Ang-1 shares the sequence homology
with fibrinogen, which binds to heparin. To investigate whether
Ang-1 binds to the ECM via its interaction with the sulfated
glycosaminoglycans, the purified Ang-1v5/His (FIG. 3A, lane 1) was
applied to a heparin-Sepharose CL-6B affinity column (Amersham
Pharmacia Biotech). The unbound flow-through was collected, and the
column was washed with 0.15, 0.3, 0.5, 1 M NaCl (FIG. 3A, lanes
3-6). The eluted fractions were collected, and along with the
flow-through fraction, they were subjected to the Western blot
analysis by using anti-v5 monoclonal antibody to detect the
presence of Ang-1v5/His in each fraction. The results indicated
that Ang-1v5/His does not bind to the heparin affinity column and
was fully recovered in the flow-through fraction (FIG. 3A, lane 2).
To confirm the above finding and avoid the possibility that the
purified soluble Ang-1v5/His may be modified and different from the
ECM-associated Ang-1 and thereby unable to bind to heparin, soluble
sulfated glycosaminoglycans, heparin, and chondroitin sulfate (200
.mu.g/ml) were added into serum-free cell culture medium (SFM) of
LLC carcinoma cells expressing Ang-1. After 2, 12, and 24 h of
incubation, the SFM and ECM fractions were collected and analyzed.
The Western blot results indicated that neither heparin nor
chondroitin sulfate releases Ang-1 from the ECM of the transfected
LLC cells (FIG. 3B, and data not shown).
[0108] In an attempt to identify the ECM component(s) that bind(s)
to Ang-1, solid phase binding assays were performed to assess the
binding affinity of Ang-1 to several ECM components. The assays
were performed in triplicate, and the results are listed in the
FIG. 4. The purified Ang-1v5 binds to whole ECM extracts derived
from LLC carcinoma cells with high affinity (FIG. 4, column 8). The
weak bindings to Matrigel, fibrinogen, and vitronectin were
observed, which can't account entirely for the high affinity
binding between Ang-1 and the ECM extracts. Ang-1 displayed no
affinity to fibronectin, laminin, collagen type I and type IV,
heparin, chondroitin sulfate, and hyaluronic acid (FIG. 4). This
result offered the possibility that Ang-1 binds to the ECM via an
unidentified ECM protein(s).
[0109] Immunocytochemistry Studies Revealed a Distinct ECM
Distribution Pattern of Angiopoietin-1--The distribution patterns
of Ang-1 and Ang-2 were investigated and compared with fibronectin,
laminin, and type I and IV collagens by performing
immunocytochemistry on LLC carcinoma cells expressing Ang-1v5 or
Ang-2v5 using anti-v5 antibody or antibodies against the
appropriate ECM proteins. The cell-free ECM deposited by LLC cells
expressing Ang-1 or Ang-2 was also examined. The
immunocytochemistry studies uncovered a distinct Ang-1 distribution
pattern in the ECM of LLC carcinoma cells, which is different from
the distribution of fibronectin, laminin, and types I and IV
collagen (FIGS. 5, A and B). Ang-1 is incorporated into the ECM as
small granule-like depositions, which are more or less evenly
distributed beneath the cells (FIGS. 5A, a and b). Some cell-free
spaces, which are left behind by the migrating cells, are positive
for the similar Ang-1 depositions (FIG. 5A, a, arrow). The
distribution pattern of Ang-1 was preserved even after the cells
were lifted by the treatment of EDTA (FIG. 5A, b), which indicated
that instead of loosely binding to the tumor cells, those Ang-1
proteins are incorporated into the ECM of the tumor cells. On the
contra, there is no trace of Ang-2 in the cell-free ECM deposited
by the cells expressing Ang-2 (FIG. 5A, d). Ang-2 was only detected
in the cytoplasm of the transfected cells, which presumably
reflects the presence of Ang-2 in the secretory pathway of the
cells (FIG. 5A, c, arrowheads).
[0110] Angiopoietin-1 Is Released in the Response to Phorbol
12-Myristate 13-Acetate (PMA)--The association of Ang-1 with the
ECM of tumor cells led us to explore the regulatory mechanisms of
its incorporation and releasing, which may modulate its activity.
The confluent LLC carcinoma cells expressing Ang-1 were cultured
overnight in the presence of different growth factors or PMA in
serum-free cell culture medium. After releasing the cells from the
culture dishes, the remaining ECM components were extracted with
1.times.SDS Laemmli buffer and subjected to Western blot analysis
using anti-v5 antibody. The results indicated that PMA stimulates
the releasing of the ECM-associated Ang-1 (FIG. 6, lane 2).
Transforming growth factor-1 (TGF-1) promotes slightly the
incorporation of Ang-1 into the ECM, which may reflect the positive
effect of TGF-1 on synthesis of the ECM components (FIG. 6, lane
3).
[0111] The ECM-associated Ang-1 Does Not Bind to Tie-2-Fc Fusion
Protein--To determine whether the ECM-associated Ang-1 proteins
bind to Tie-2-Fc fusion proteins, the confluent LLC cells
expressing Ang-1 or the ECM deposited by the LLC cells expressing
Ang-1 were fixed and incubated with anti-v5 antibody or the
purified Tie-2-Fc fusion proteins. Anti-v5 antibody and
FITC-conjugated rabbit anti-mouse antibody (Sigma) revealed that
many Ang-1 proteins were deposited into the ECM of the tumor cells
(FIGS. 7B, a and b); however, Ang-1 proteins in the ECM exhibited
no affinity to Tie-2-Fc fusion proteins (FIGS. 7B, c and d). In
order to eliminate the possibility that the fixation procedure
could affect the binding between the ECM-associated Ang-1 and
Tie-2-Fc fusion protein, Tie-2-Fc fusion proteins were directly
added into the cultured LLC carcinoma cells expressing Ang-1 or
into the ECM freshly derived from the LLC cells expressing Ang-1.
No binding was detected in both cases (data not shown). Those
ECM-associated Ang-1 proteins were extracted by 2 M urea/Tris-HCl
buffer (pH 7.4), dialyzed in PBS, and immunoprecipitated using
Tie-2-Fc fusion proteins and protein A beads. The precipitated
proteins were subjected to Western blot analysis using anti-v5
antibody. Ang-1 proteins were found to be precipitated by Tie-2-Fc
fusion proteins (FIG. 7A, lane 2). Together, those results
suggested that after their secretion from the tumor cells, Ang-1
proteins are incorporated into the ECM, and their Tie-2-binding
sites are no longer accessible; upon the release from the ECM, the
solubilized Ang-1 proteins regained their ability to bind to Tie-2.
Under physiologic conditions, this may serve as an efficient
regulatory mechanism for Ang-1 activity.
[0112] Tie-2 Phosphorylation Is Achieved by Adhering HUVECs to the
ECM Containing Ang-1 Proteins--To study whether the ECM-associated
Ang-1 plays a role in angiogenesis, the ability of the
ECM-associated Ang-1 to promote Tie-2 receptor phosphorylation was
evaluated using HUVECs. The subconfluent HUVECs were serum-starved
for about 14 h and then lifted from the culture dishes. 1.times.106
cells were seeded onto a plastic culture dish with (FIG. 8, lane 3)
or without (FIG. 8, lane 1) purified soluble Ang-1 (200 ng/ml) or
the culture dish containing the ECM components deposited by the
confluent LLC cells expressing Ang-1 (FIG. 8, lane 4) or Ang-2
(FIG. 8, lane 2). The HUVECs were incubated on those cell culture
dishes for 30 ml and then lysed at 4.degree. C. with the lysis
buffer. The amount of the Tie-2 proteins in the lysates was
determined by Western blot analysis of 50 .mu.g of proteins from
each lysate using anti-Tie-2 antibody (C-20, Santa Cruz
Biotechnology, FIG. 8B). The results indicated that the endothelial
cells used in the experiments express a similar amount of Tie-2
receptors (FIG. 8B). The phosphorylated Tie-2 proteins in the
lysates were assessed by performing immunoprecipitation using
anti-Tie-2 polyclonal antibody (C-20, Santa Cruz Biotechnology) and
analyzing the precipitated proteins on Western blot using
anti-phosphotyrosine antibody (Y20, Calbiochem). The results
indicated that Tie-2 receptors on HUVECs are phosphorylated by
Ang-1 derived from the ECM, and the phosphorylation is inhibited by
the addition of the excess amount of Tie-2-Fc fusion proteins (2
.mu.g, FIG. 8, lane 5).
[0113] To assess whether HUVECs release Ang-1 proteins from the
ECM, HUVECs cells (1.times.106 cells/100-mm dish) or the serum-free
culture media (SFM) alone were placed on the ECM deposited by LLC
cells expressing Ang-1 for 30 min. The cell culture dishes with or
without HUVECs were treated with 5 mM EDTA in PBS for 5 min to
release the adhering cells. The remaining ECM materials on the
culture dishes were extracted and subjected to Western blot
analysis, and the results indicated that the reduced amount of
Ang-1 is retained in the ECM after the adhesion of HUVECs compared
with that retained in the ECM incubated with SFM alone (data not
shown). Thus, HUVECs promote the release of Ang-1 from the ECM.
[0114] The Domain That Mediates the ECM Binding of Ang-1 Is Mapped
to Its Linker Peptide Region--To determine which domain of Ang-1
mediates its ECM association, several expression constructs were
made as indicated in FIG. 9A. They were the coiled-coil domain, the
coiled-coil plus linker peptide region, and the FHD of Ang-1. The
cDNA sequence encoding the signal peptide of Ang-1 was constructed
into the N terminus of the above cDNA fragments so those fragments
can be secreted properly (FIG. 9A). The full-length Ang-1 and -2
and the fragments of Ang-1, which contain C-terminal v5 epitope
tags, were used to transfect COS-7 cells. 72 h after the transient
transfection, the cell culture supernatants and the ECM materials
derived from the transfected cells were either collected or
extracted from the cell culture dishes and subjected to Western
blot analyses using anti-v5 antibody to determine the distribution
patterns of Ang-1, Ang-2, and the different fragments of Ang-1. The
results indicated that the linker peptide region of Ang-1 between
the coiled-coil and the fibrinogen-like domains, which contains 27
amino acids, is mainly responsible for the ECM association of Ang-1
(FIGS. 9, B and C, lanes 5 and 6). A weak interaction between the
coiled-coil domain of Ang-1 and the ECM was also detected (FIGS. 9,
B and C, lanes 7 and 8).
[0115] To investigate whether the ECM binding domain in the linker
peptide region of Ang-1 is also present in Ang-2, we first compared
the sequence homology between the domains of Ang-1 and Ang-2. We
found that the percentages of the identical amino acids are 59, 19,
and 64%, respectively, in the coiled-coil domain, the linker
peptide region, and the fibrinogen-like domains of Ang-1 and Ang-2.
No significant homology in the linker peptide region implied that
the ECM binding domain in this region of Ang-1 is likely absent in
the Ang-2 molecule.
[0116] To confirm that the absence of the ECM binding of Ang-2 is
due to the lack of the ECM-binding site(s) in the molecule, but not
the blockage of the binding site(s) by possible steric restraints
in the full-length Ang-2 molecule, we made three additional
deletions of Ang-2, which are similar to those of Ang-1 deletions
(FIG. 9A). They are the coiled-coil domain, the coiled-coil plus
the linker peptide region, and the fibrinogen-like domain of Ang-2.
All the deletion constructs contain the N-terminal signal peptides
of Ang-2 for their proper expression and secretion and the
C-terminal v5 epitope tags derived from the pEF/6/v5-His expression
vectors for their detection. After confirming the authenticities of
the expression constructs by DNA sequencing, they were used to
transfect COS-7 cells. After the transient transfection, the
distributions of Ang-2 fragments in the cell culture media and the
ECM fractions were examined by Western blot analysis. Our results
indicated that all three fragments of Ang-2, the coiled-coil
domain, the coiled-coil plus the linker peptide region, and the
fibrinogen-like domain, are secreted as the full-length Ang-2; none
of them associates with the ECM of the transfected COS-7 (data not
shown). This result indicated clearly that unlike Ang-1, Ang-2 does
not contain the ECM binding domain.
Discussion
[0117] Studies have shown that angiopoietin-1 and -2 are unique
antagonists. Ang-2 blocks tyrosine phosphorylation of Tie-2 induced
by Ang-1 and disrupts angiogenesis in vivo. Ang-1 and -2 play
different roles in tumor angiogenesis. Ang-2 inhibits tumor
angiogenesis by blocking recruitment of smooth muscle cells to the
newly formed blood vessels and causing apoptosis to the endothelial
cells.
[0118] Functional Significance of the ECM Association of Ang-1--In
this study, we examined the potential relationship of Ang-1 and -2
with the ECM, which is known to modulate activities of many growth
factors, including VEGF and bFGF. Our results indicated that unlike
Ang-2, Ang-1 is secreted and incorporated into and sequestered by
the ECM. The different ECM association capacity enables those two
antagonists to regulate local and distant angiogenesis differently.
Ang-1 is most likely to regulate angiogenesis in the vicinity of
its secretion sites, whereas Ang-2 is likely to diffuse through
interstitial tissues and blood vessels to the distant organs.
[0119] The ability to distribute Ang-1 and -2 to local environment
and/or distant sites may play an important role in regulating tumor
dormancy in some situations. Both Ang-1 and -2 are expressed by
endothelial and tumor cells. During the growth of primary tumors,
balanced expression of local Ang-1 and -2 and expression of other
pro-angiogenic factors, such as VEGF, may ensure tumor angiogenesis
and growth in the primary sites. When Tie-2 receptors on local
endothelial cells are saturated, it is likely that the excess
amount of Ang-1 produced by the primary tumors would be
incorporated into and sequestered by the surrounding ECM, whereas
the excessive amount of Ang-2 may diffuse to distant organs. The
micrometastases seeded and developed in the distant organs, such as
lung and liver, have microenvironments that contain pro- and
anti-angiogenic factors produced by local micrometastatic tumors
and surrounding host stromal tissues and are more or less similar
to the microenvironments of primary tumors. However, on top of the
microenvironments, Ang-2, not Ang-1, produced by the predominant
primary tumors, which is stable and has a long half-life (data not
shown), could travel to the distant organs and change the balance
in favor of inhibiting tumor angiogenesis, which may contribute to
the dormancy observed in some secondary tumors.
[0120] The ECM Association of Ang-1 Provides a Different Regulatory
Mechanism for the Availability of Ang-1--Ang-1 is wildly expressed
both in embryo and in adult and exhibits a more uniform expression
pattern compared with that of Ang-2 Ang-2 has a wild expression
pattern in embryo. However, in adult tissues, its expression is
restricted in the tissues where vascular remodeling is ongoing,
such as ovary, uterus, and placenta. Ang-2 is up-regulated by
hypoxia and several different cytokines including VEGF and tumor
necrosis factor, whereas the expression of Ang-1 is not
affected.
[0121] The association of Ang-1 with the ECM reported herein
offered a different type of regulation of the availability of
Ang-1. Instead of regulating its production, as Ang-2, free Ang-1
in microenvironment can be regulated by changing its ECM
association status. The factors that promote or inhibit its ECM
incorporation or releasing affect the interaction between Ang-1 and
Tie-2 receptor, sequential signal transduction, and angiogenesis.
This type of regulation provides a quicker response to the changes
in the microenvironment without the requirement of mRNA and
proteins syntheses. It is well established that the activities of
some growth factors are regulated in that way, including TGF.
[0122] The Biochemical Characters of the ECM Association of
Ang-1--An extensive effort was made to try to identify the ECM
component(s) that mediate(s) the ECM association of Ang-1.
Matrigel, fibrinogen, and to a less extent vitronectin exhibited
weak affinities to Ang-1, which is much lower than that of
unpurified ECM extracts derived from LLC cells and cannot account
entirely for the ECM association of Ang-1 (FIG. 4). The
immunocytochemistry studios indicated that the ECM distribution
pattern of Ang-1 is unique and unlike that of fibronectin, laminin,
and collagen types I and IV (FIG. 5).
[0123] To determine the effect of the ECM association on the
function of Ang-1, we assessed whether Tie-2-Fc fusion protein
hinds to the ECM-associated Ang-1. The result indicated that the
ECM-associated Ang-1 is not accessible for Tie-2, and the ECM
serves as a storage and sequestration site for Ang-1. The
incubation of HUVECs on the ECM containing Ang-1 induced the
release of Ang-1 from the ECM and tyrosine phosphorylation of
Tie-2, which indicated that HUVECs cells are able to respond to
Ang-1 originally sequestered in the ECM (FIG. 8). This may reflect
the in vivo situations and indicate that activity of Ang-1 is
restricted in local environment and regulated more tightly than
that of Ang-2.
[0124] The Domain of Ang-1 That Mediates Its ECM Association Is the
Linker Peptide Region--The deletion analysis indicated that the
linker peptide region of Ang-1 between the coiled-coil domain and
the FHD mediates the ECM association of Ang-1 (FIG. 9). The
identification of the ECM association domain of Ang-1 will allow us
to study the importance of the ECM association on Ang-1 function in
the future.
[0125] In summary, we have discovered that Ang-1 is incorporated
into the ECM after its secretion, whereas Ang-2 is secreted and
does not associate with the ECM. The domain that mediates the ECM
association of Ang-1 is mapped to the linker peptide region between
the coiled-coil and the fibrinogen homology domains. The different
ECM association capacity of Ang-1 and Ang-2 offers a possible
mechanism for the distinct regulations of local and distant tumor
angiogenesis by two antagonistic factors.
ABBREVIATIONS
[0126] The abbreviations used are: ECM, extracellular matrix;
Ang-1, angiopoietin-1; Ang-2, angiopoietin-2; TA3, TA3 murine
mammary carcinoma; LLC, Lewis lung carcinoma; HUVEC, human
umbilical vein endothelial cell; FHD, fibrinogen homology domain;
PBS, phosphate-buffered saline solution; BSA, bovine serum albumin;
PCR, polymerase chain reaction; RT-PCR, reverse
transcriptase-polymerase chain reaction; FITC, fluorescein
isothiocyanate; DOC, deoxycholate; SFM, serum-free medium; PMA,
phorbol 12-myristate 13-acetate; bFGF, basic fibroblast growth
factor; TGF-, transforming growth factor-; VEGF, vascular
endothelial growth factor.
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Sequence CWU 1
1
19126PRTMus musculus 1Val His Asn Leu Val Ser Leu Cys Thr Lys Glu
Gly Val Leu Leu Lys1 5 10 15Gly Gly Lys Arg Glu Glu Glu Lys Pro
Phe20 25226PRThomo sapiens 2Val His Asn Leu Val Asn Leu Cys Thr Lys
Glu Gly Val Leu Leu Lys1 5 10 15Gly Gly Lys Arg Glu Glu Glu Lys Pro
Phe20 2532149DNAHomo sapiens 3cagctgactc aggcaggctc catgctgaac
ggtcacacag agaggaaaca ataaatctca 60gctactatgc aataaatatc tcaagtttta
acgaagaaaa acatcattgc agtgaaataa 120aaaattttaa aattttagaa
caaagctaac aaatggctag ttttctatga ttcttcttca 180aacgctttct
ttgaggggga aagagtcaaa caaacaagca gttttacctg aaataaagaa
240ctagttttag aggtcagaag aaaggagcaa gttttgcgag aggcacggaa
ggagtgtgct 300ggcagtacaa tgacagtttt cctttccttt gctttcctcg
ctgccattct gactcacata 360gggtgcagca atcagcgccg aagtccagaa
aacagtggga gaagatataa ccggattcaa 420catgggcaat gtgcctacac
tttcattctt ccagaacacg atggcaactg tcgtgagagt 480acgacagacc
agtacaacac aaacgctctg cagagagatg ctccacacgt ggaaccggat
540ttctcttccc agaaacttca acatctggaa catgtgatgg aaaattatac
tcagtggctg 600caaaaacttg agaattacat tgtggaaaac atgaagtcgg
agatggccca gatacagcag 660aatgcagttc agaaccacac ggctaccatg
ctggagatag gaaccagcct cctctctcag 720actgcagagc agaccagaaa
gctgacagat gttgagaccc aggtactaaa tcaaacttct 780cgacttgaga
tacagctgct ggagaattca ttatccacct acaagctaga gaagcaactt
840cttcaacaga caaatgaaat cttgaagatc catgaaaaaa acagtttatt
agaacataaa 900atcttagaaa tggaaggaaa acacaaggaa gagttggaca
ccttaaagga agagaaagag 960aaccttcaag gcttggttac tcgtcaaaca
tatataatcc aggagctgga aaagcaatta 1020aacagagcta ccaccaacaa
cagtgtcctt cagaagcagc aactggagct gatggacaca 1080gtccacaacc
ttgtcaatct ttgcactaaa gaaggtgttt tactaaaggg aggaaaaaga
1140gaggaagaga aaccatttag agactgtgca gatgtatatc aagctggttt
taataaaagt 1200ggaatctaca ctatttatat taataatatg ccagaaccca
aaaaggtgtt ttgcaatatg 1260gatgtcaatg ggggaggttg gactgtaata
caacatcgtg aagatggaag tctagatttc 1320caaagaggct ggaaggaata
taaaatgggt tttggaaatc cctccggtga atattggctg 1380gggaatgagt
ttatttttgc cattaccagt cagaggcagt acatgctaag aattgagtta
1440atggactggg aagggaaccg agcctattca cagtatgaca gattccacat
aggaaatgaa 1500aagcaaaact ataggttgta tttaaaaggt cacactggga
cagcaggaaa acagagcagc 1560ctgatcttac acggtgctga tttcagcact
aaagatgctg ataatgacaa ctgtatgtgc 1620aaatgtgccc tcatgttaac
aggaggatgg tggtttgatg cttgtggccc ctccaatcta 1680aatggaatgt
tctatactgc gggacaaaac catggaaaac tgaatgggat aaagtggcac
1740tacttcaaag ggcccagtta ctccttacgt tccacaacta tgatgattcg
acctttagat 1800ttttgaaagc gcaatgtcag aagcgattat gaaagcaaca
aagaaatccg gagaagctgc 1860caggtgagaa actgtttgaa aacttcagaa
gcaaacaata ttgtctccct tccagcaata 1920agtggtagtt atgtgaagtc
accaaggttc ttgaccgtga atctggagcc gtttgagttc 1980acaagagtct
ctacttgggg tgacagtgct cacgtggctc gactatagaa aactccactg
2040actgtcgggc tttaaaaagg gaagaaactg ctgagcttgc tgtgcttcaa
actactactg 2100gaccttattt tggaactatg gtagccagat gataaatatg
gttaatttc 21494498PRTHomo sapiens 4Met Thr Val Phe Leu Ser Phe Ala
Phe Leu Ala Ala Ile Leu Thr His1 5 10 15Ile Gly Cys Ser Asn Gln Arg
Arg Ser Pro Glu Asn Ser Gly Arg Arg20 25 30Tyr Asn Arg Ile Gln His
Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro35 40 45Glu His Asp Gly Asn
Cys Arg Glu Ser Thr Thr Asp Gln Tyr Asn Thr50 55 60Asn Ala Leu Gln
Arg Asp Ala Pro His Val Glu Pro Asp Phe Ser Ser65 70 75 80Gln Lys
Leu Gln His Leu Glu His Val Met Glu Asn Tyr Thr Gln Trp85 90 95Leu
Gln Lys Leu Glu Asn Tyr Ile Val Glu Asn Met Lys Ser Glu Met100 105
110Ala Gln Ile Gln Gln Asn Ala Val Gln Asn His Thr Ala Thr Met
Leu115 120 125Glu Ile Gly Thr Ser Leu Leu Ser Gln Thr Ala Glu Gln
Thr Arg Lys130 135 140Leu Thr Asp Val Glu Thr Gln Val Leu Asn Gln
Thr Ser Arg Leu Glu145 150 155 160Ile Gln Leu Leu Glu Asn Ser Leu
Ser Thr Tyr Lys Leu Glu Lys Gln165 170 175Leu Leu Gln Gln Thr Asn
Glu Ile Leu Lys Ile His Glu Lys Asn Ser180 185 190Leu Leu Glu His
Lys Ile Leu Glu Met Glu Gly Lys His Lys Glu Glu195 200 205Leu Asp
Thr Leu Lys Glu Glu Lys Glu Asn Leu Gln Gly Leu Val Thr210 215
220Arg Gln Thr Tyr Ile Ile Gln Glu Leu Glu Lys Gln Leu Asn Arg
Ala225 230 235 240Thr Thr Asn Asn Ser Val Leu Gln Lys Gln Gln Leu
Glu Leu Met Asp245 250 255Thr Val His Asn Leu Val Asn Leu Cys Thr
Lys Glu Gly Val Leu Leu260 265 270Lys Gly Gly Lys Arg Glu Glu Glu
Lys Pro Phe Arg Asp Cys Ala Asp275 280 285Val Tyr Gln Ala Gly Phe
Asn Lys Ser Gly Ile Tyr Thr Ile Tyr Ile290 295 300Asn Asn Met Pro
Glu Pro Lys Lys Val Phe Cys Asn Met Asp Val Asn305 310 315 320Gly
Gly Gly Trp Thr Val Ile Gln His Arg Glu Asp Gly Ser Leu Asp325 330
335Phe Gln Arg Gly Trp Lys Glu Tyr Lys Met Gly Phe Gly Asn Pro
Ser340 345 350Gly Glu Tyr Trp Leu Gly Asn Glu Phe Ile Phe Ala Ile
Thr Ser Gln355 360 365Arg Gln Tyr Met Leu Arg Ile Glu Leu Met Asp
Trp Glu Gly Asn Arg370 375 380Ala Tyr Ser Gln Tyr Asp Arg Phe His
Ile Gly Asn Glu Lys Gln Asn385 390 395 400Tyr Arg Leu Tyr Leu Lys
Gly His Thr Gly Thr Ala Gly Lys Gln Ser405 410 415Ser Leu Ile Leu
His Gly Ala Asp Phe Ser Thr Lys Asp Ala Asp Asn420 425 430Asp Asn
Cys Met Cys Lys Cys Ala Leu Met Leu Thr Gly Gly Trp Trp435 440
445Phe Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Phe Tyr Thr
Ala450 455 460Gly Gln Asn His Gly Lys Leu Asn Gly Ile Lys Trp His
Tyr Phe Lys465 470 475 480Gly Pro Ser Tyr Ser Leu Arg Ser Thr Thr
Met Met Ile Arg Pro Leu485 490 495Asp Phe52044DNAMus musculus
5ctgacgcggg caggctccac gctgaacggt tacacagaga ggaaacaata aatctaagct
60actattgcaa taaatatctc aagttttaac gaaggaaact atcattacag ttaaaatttt
120ttaaagtaac gcttttttag aacaaagcta acaaatggct agttttctgt
ggatcttctt 180caaacgcttt ctttaacggg gaaagagtca aacaagcagt
tttacctgaa ataaagaact 240agtttaaagg tcagaagaga agagcaagct
ttgcaggagg cacggaaggc aagcgctggc 300agtacaatga cagttttcct
ttcctttgca ttcttcgctg ccattctgac tcacataggg 360tgcagcaacc
agcgccgaaa tccagaaaac ggagggagaa gatataaccg gattcaacat
420gggcaatgtg cctacacttt cattcttcca gaacacgacg ggaactgccg
tgagagtgcg 480acagagcagt acaacaccaa cgctctgcaa agggatgctc
cacacgtgga gccggatttc 540tcttcccaga aacttcagca tctggagcat
gtgatggaaa attatactca gtggctgcaa 600aaacttgaga attacattgt
ggaaaatatg aagtcggaga tggcccagat acaacagaat 660gctgttcaaa
accacacggc caccatgctt gagataggaa ccagtctctt atctcagact
720gcagagcaga cccgaaagct gacagatgtt gagacccagg tactaaatca
aacatcccga 780cttgaaatac aactgctaga gaattcatta tcaacataca
agctagagaa gcaacttctc 840caacagacaa atgaaattct gaagattcac
gaaaaaaaca gtttactaga gcacaaaatc 900ttagaaatgg agggaaaaca
caaagaagaa ttggacacct tgaaggagga gaaagaaaac 960cttcaaggct
tggtttctcg tcagacattc atcatccagg agttggagaa gcaacttagt
1020agagctacca acaacaacag catcctgcag aagcaacaac tggagctcat
ggacacagtt 1080cataaccttg tcagcctttg cactaaagaa ggtgttttgc
taaagggagg aaaaagagaa 1140gaagagaaac catttcgaga ctgtgcagat
gtatatcaag ctggttttaa taaaagtgga 1200atctacacta tttattttaa
taatatgcca gaacccaaaa aggtattttg caatatggat 1260gtgaatgggg
gaggttggac agtaatacaa caccgggaag atggaagcct ggatttccag
1320aggggctgga aggagtataa aatgggtttt gggaatccct ctggtgaata
ttggctcggg 1380aacgagttca tttttgcaat aaccagtcag aggcagtaca
tgctgaggat tgagctgatg 1440gactgggaag ggaaccgagc ctactcacag
tacgacagat tccacatagg aaatgaaaag 1500cagaactata ggttatattt
aaaaggtcac acagggacag caggcaaaca gagcagcttg 1560atcttacacg
gtgctgattt cagcacgaag gatgctgata acgacaactg tatgtgcaaa
1620tgcgctctca tgctaacagg aggttggtgg ttcgatgcct gtggcccttc
caatctaaat 1680ggaatgttct acactgcggg acaaaatcat ggaaaactga
atgggataaa gtggcactac 1740ttcaaagggc ccagttactc cttacgttcc
accaccatga tgatccggcc cttggacttt 1800tgaaggtgct atgccagtat
tagaaagctg caaagaaagc tgggcatgtt cccagatgag 1860aagctagtca
gaggcttcag aaacaaccaa cattgtctcc gttccagcag caagtggtta
1920tgtcatgtca cctgggtact taacaatgga tttggagcct tctgaggtca
acagaatcgc 1980cacttgggtc cagagaatgc cactcacaat catgtttaaa
agggaagaaa cttctcagct 2040tgct 20446498PRTMus musculus 6Met Thr Val
Phe Leu Ser Phe Ala Phe Phe Ala Ala Ile Leu Thr His1 5 10 15Ile Gly
Cys Ser Asn Gln Arg Arg Asn Pro Glu Asn Gly Gly Arg Arg20 25 30Tyr
Asn Arg Ile Gln His Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro35 40
45Glu His Asp Gly Asn Cys Arg Glu Ser Ala Thr Glu Gln Tyr Asn Thr50
55 60Asn Ala Leu Gln Arg Asp Ala Pro His Val Glu Pro Asp Phe Ser
Ser65 70 75 80Gln Lys Leu Gln His Leu Glu His Val Met Glu Asn Tyr
Thr Gln Trp85 90 95Leu Gln Lys Leu Glu Asn Tyr Ile Val Glu Asn Met
Lys Ser Glu Met100 105 110Ala Gln Ile Gln Gln Asn Ala Val Gln Asn
His Thr Ala Thr Met Leu115 120 125Glu Ile Gly Thr Ser Leu Leu Ser
Gln Thr Ala Glu Gln Thr Arg Lys130 135 140Leu Thr Asp Val Glu Thr
Gln Val Leu Asn Gln Thr Ser Arg Leu Glu145 150 155 160Ile Gln Leu
Leu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln165 170 175Leu
Leu Gln Gln Thr Asn Glu Ile Leu Lys Ile His Glu Lys Asn Ser180 185
190Leu Leu Glu His Lys Ile Leu Glu Met Glu Gly Lys His Lys Glu
Glu195 200 205Leu Asp Thr Leu Lys Glu Glu Lys Glu Asn Leu Gln Gly
Leu Val Ser210 215 220Arg Gln Thr Phe Ile Ile Gln Glu Leu Glu Lys
Gln Leu Ser Arg Ala225 230 235 240Thr Asn Asn Asn Ser Ile Leu Gln
Lys Gln Gln Leu Glu Leu Met Asp245 250 255Thr Val His Asn Leu Val
Ser Leu Cys Thr Lys Glu Gly Val Leu Leu260 265 270Lys Gly Gly Lys
Arg Glu Glu Glu Lys Pro Phe Arg Asp Cys Ala Asp275 280 285Val Tyr
Gln Ala Gly Phe Asn Lys Ser Gly Ile Tyr Thr Ile Tyr Phe290 295
300Asn Asn Met Pro Glu Pro Lys Lys Val Phe Cys Asn Met Asp Val
Asn305 310 315 320Gly Gly Gly Trp Thr Val Ile Gln His Arg Glu Asp
Gly Ser Leu Asp325 330 335Phe Gln Arg Gly Trp Lys Glu Tyr Lys Met
Gly Phe Gly Asn Pro Ser340 345 350Gly Glu Tyr Trp Leu Gly Asn Glu
Phe Ile Phe Ala Ile Thr Ser Gln355 360 365Arg Gln Tyr Met Leu Arg
Ile Glu Leu Met Asp Trp Glu Gly Asn Arg370 375 380Ala Tyr Ser Gln
Tyr Asp Arg Phe His Ile Gly Asn Glu Lys Gln Asn385 390 395 400Tyr
Arg Leu Tyr Leu Lys Gly His Thr Gly Thr Ala Gly Lys Gln Ser405 410
415Ser Leu Ile Leu His Gly Ala Asp Phe Ser Thr Lys Asp Ala Asp
Asn420 425 430Asp Asn Cys Met Cys Lys Cys Ala Leu Met Leu Thr Gly
Gly Trp Trp435 440 445Phe Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly
Met Phe Tyr Thr Ala450 455 460Gly Gln Asn His Gly Lys Leu Asn Gly
Ile Lys Trp His Tyr Phe Lys465 470 475 480Gly Pro Ser Tyr Ser Leu
Arg Ser Thr Thr Met Met Ile Arg Pro Leu485 490 495Asp
Phe71891DNARattus norvegicus 7aggctccacg ctgaacggtt acacagagag
gaaacaataa atctaagcta ctattgcaat 60aaatatctca agttttaacg aaggaaacta
tcattacagt aattttttta agtaacgcgt 120tttataacaa agctaacaaa
tggctagttt tctgtggatc ttcttcaaat gctttcttta 180acggggagag
cgtcaaacaa ccagttttac ctgaaataaa gaactagttt aaaggtcaga
240agagaggagc aagctttgca ggaggcacgg aaggcgagtg ctggcagtac
aatgacagtt 300ttcctttcct ttgcattctt cgctgccatt ctgactcaca
tagggtgcag caaccagcgc 360cggagtccag aaaacggagg gagaagatat
aaccgaattc aacatgggca atgtgcctac 420actttcattc ttccagaaca
cgacgggaac tgccgtgaga gtgcgacaga gcagtacaac 480accaacgctc
tgcaaaggga tgctccacac gtggagacgg atttctcttc ccagaaactt
540cagcatctgg agcatgtgat ggaaaattat actcagtggc tgcaaaaact
tgagaattac 600attgtggaaa atatgaagtc ggagatggcc cagatacaac
agaatgcggt tcaaaaccac 660acggccacca tgctggagat aggaaccagc
ctcttgtctc agactgcaga gcagacccga 720aagctcacag atgtggagac
ccaggtacta aatcaaacat cccgtcttga aatccaactg 780ctggagaatt
cattatcaac atacgagcta gagaaacagc ttctccaaca gacaaatgaa
840attctgaaga ttcaggaaaa aaacagttta ttagagcata aaatcctaga
aatggaggga 900aaacacaagg aagagctgga caccttgaag gaggagaaag
aaaaccttca aggcttggtt 960actcgtcaga cattcatcat ccaagaattg
gagaagcaac ttagcagagc taccagcaac 1020aacagtgttc tgcagaagca
acaactggag ctcatggaca cagtccataa ccttgtcagc 1080ctttgcacaa
aagaagtttt gctaaaggga ggaaaaagag aagaagagaa accatttcga
1140gactgtgcag atgtatatca agctggtttt aataagagtg gaatctacac
tatttatttt 1200aataatatgc cagaacccaa aaaggtattt tgcaatatgg
atgtgaatga aggaggatgg 1260acagtaatac aacaccgtga ggatggaagc
ctagatttcc agaggggctg gaaggagtat 1320aaaatgggtt ttgggaatcc
ctctggtgaa tattggcttg ggaacgagtt catttttgca 1380ataaccagtc
agaggcagta catgctgagg atcgagctga tggactggga agggaaccga
1440gcctactcac agtacgacag attccacata ggaaaccaga agcagaacta
caggttatat 1500ttaaagggtc acacggggac agcaggcaaa cagagcagct
tgatcttaca tggtgctgat 1560ttcagcacaa aggacgctga taacgacaac
tgtatgtgca aatgcgccct tatgctaaca 1620ggaggttggt ggtttgatgc
ctgtggccct tccaatctaa acggaatgtt ctacactgca 1680gggcaaaacc
atggaaaact gaatgggata aagtggcact acttcaaagg acccagttac
1740tccttacgtt ccacaaccat gatgatccgg cccttggact tctgaaggcg
ctatgcctag 1800tattagaaac ctgaaataaa tctggggatg ttcccgaatg
agaagctatc tggaagcttc 1860cgaaacaacc cagcattgtc tccgttccag c
18918497PRTRattus norvegicus 8Met Thr Val Phe Leu Ser Phe Ala Phe
Phe Ala Ala Ile Leu Thr His1 5 10 15Ile Gly Cys Ser Asn Gln Arg Arg
Ser Pro Glu Asn Gly Gly Arg Arg20 25 30Tyr Asn Arg Ile Gln His Gly
Gln Cys Ala Tyr Thr Phe Ile Leu Pro35 40 45Glu His Asp Gly Asn Cys
Arg Glu Ser Ala Thr Glu Gln Tyr Asn Thr50 55 60Asn Ala Leu Gln Arg
Asp Ala Pro His Val Glu Thr Asp Phe Ser Ser65 70 75 80Gln Lys Leu
Gln His Leu Glu His Val Met Glu Asn Tyr Thr Gln Trp85 90 95Leu Gln
Lys Leu Glu Asn Tyr Ile Val Glu Asn Met Lys Ser Glu Met100 105
110Ala Gln Ile Gln Gln Asn Ala Val Gln Asn His Thr Ala Thr Met
Leu115 120 125Glu Ile Gly Thr Ser Leu Leu Ser Gln Thr Ala Glu Gln
Thr Arg Lys130 135 140Leu Thr Asp Val Glu Thr Gln Val Leu Asn Gln
Thr Ser Arg Leu Glu145 150 155 160Ile Gln Leu Leu Glu Asn Ser Leu
Ser Thr Tyr Glu Leu Glu Lys Gln165 170 175Leu Leu Gln Gln Thr Asn
Glu Ile Leu Lys Ile Gln Glu Lys Asn Ser180 185 190Leu Leu Glu His
Lys Ile Leu Glu Met Glu Gly Lys His Lys Glu Glu195 200 205Leu Asp
Thr Leu Lys Glu Glu Lys Glu Asn Leu Gln Gly Leu Val Thr210 215
220Arg Gln Thr Phe Ile Ile Gln Glu Leu Glu Lys Gln Leu Ser Arg
Ala225 230 235 240Thr Ser Asn Asn Ser Val Leu Gln Lys Gln Gln Leu
Glu Leu Met Asp245 250 255Thr Val His Asn Leu Val Ser Leu Cys Thr
Lys Glu Val Leu Leu Lys260 265 270Gly Gly Lys Arg Glu Glu Glu Lys
Pro Phe Arg Asp Cys Ala Asp Val275 280 285Tyr Gln Ala Gly Phe Asn
Lys Ser Gly Ile Tyr Thr Ile Tyr Phe Asn290 295 300Asn Met Pro Glu
Pro Lys Lys Val Phe Cys Asn Met Asp Val Asn Glu305 310 315 320Gly
Gly Trp Thr Val Ile Gln His Arg Glu Asp Gly Ser Leu Asp Phe325 330
335Gln Arg Gly Trp Lys Glu Tyr Lys Met Gly Phe Gly Asn Pro Ser
Gly340 345 350Glu Tyr Trp Leu Gly Asn Glu Phe Ile Phe Ala Ile Thr
Ser Gln Arg355 360 365Gln Tyr Met Leu Arg Ile Glu Leu Met Asp Trp
Glu Gly Asn Arg Ala370 375 380Tyr Ser Gln Tyr Asp Arg Phe His Ile
Gly Asn Gln Lys Gln Asn Tyr385 390 395 400Arg Leu Tyr Leu Lys Gly
His Thr Gly Thr Ala Gly Lys Gln Ser Ser405 410 415Leu Ile Leu His
Gly Ala Asp Phe Ser Thr Lys Asp Ala Asp Asn Asp420 425 430Asn Cys
Met Cys Lys Cys Ala Leu Met Leu Thr Gly Gly Trp Trp Phe435 440
445Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Phe Tyr Thr Ala
Gly450 455 460Gln Asn His Gly Lys Leu Asn Gly Ile Lys Trp His Tyr
Phe Lys Gly465 470 475 480Pro Ser Tyr Ser Leu Arg Ser Thr Thr Met
Met Ile Arg Pro Leu Asp485
490 495Phe92269DNAHomo sapiens 9tgggttggtg tttatctcct cccagccttg
agggagggaa caacactgta ggatctgggg 60agagaggaac aaaggaccgt gaaagctgct
ctgtaaaagc tgacacagcc ctcccaagtg 120agcaggactg ttcttcccac
tgcaatctga cagtttactg catgcctgga gagaacacag 180cagtaaaaac
caggtttgct actggaaaaa gaggaaagag aagactttca ttgacggacc
240cagccatggc agcgtagcag ccctgcgttt cagacggcag cagctcggga
ctctggacgt 300gtgtttgccc tcaagtttgc taagctgctg gtttattact
gaagaaagaa tgtggcagat 360tgttttcttt actctgagct gtgatcttgt
cttggccgca gcctataaca actttcggaa 420gagcatggac agcataggaa
agaagcaata tcaggtccag catgggtcct gcagctacac 480tttcctcctg
ccagagatgg acaactgccg ctcttcctcc agcccctacg tgtccaatgc
540tgtgcagagg gacgcgccgc tcgaatacga tgactcggtg cagaggctgc
aagtgctgga 600gaacatcatg gaaaacaaca ctcagtggct aatgaagctt
gagaattata tccaggacaa 660catgaagaaa gaaatggtag agatacagca
gaatgcagta cagaaccaga cggctgtgat 720gatagaaata gggacaaacc
tgttgaacca aacagctgag caaacgcgga agttaactga 780tgtggaagcc
caagtattaa atcagaccac gagacttgaa cttcagctct tggaacactc
840cctctcgaca aacaaattgg aaaaacagat tttggaccag accagtgaaa
taaacaaatt 900gcaagataag aacagtttcc tagaaaagaa ggtgctagct
atggaagaca agcacatcat 960ccaactacag tcaataaaag aagagaaaga
tcagctacag gtgttagtat ccaagcaaaa 1020ttccatcatt gaagaactag
aaaaaaaaat agtgactgcc acggtgaata attcagttct 1080tcaaaagcag
caacatgatc tcatggagac agttaataac ttactgacta tgatgtccac
1140atcaaactca gctaaggacc ccactgttgc taaagaagaa caaatcagct
tcagagactg 1200tgctgaagta ttcaaatcag gacacaccac aaatggcatc
tacacgttaa cattccctaa 1260ttctacagaa gagatcaagg cctactgtga
catggaagct ggaggaggcg ggtggacaat 1320tattcagcga cgtgaggatg
gcagcgttga ttttcagagg acttggaaag aatataaagt 1380gggatttggt
aacccttcag gagaatattg gctgggaaat gagtttgttt cgcaactgac
1440taatcagcaa cgctatgtgc ttaaaataca ccttaaagac tgggaaggga
atgaggctta 1500ctcattgtat gaacatttct atctctcaag tgaagaactc
aattatagga ttcaccttaa 1560aggacttaca gggacagccg gcaaaataag
cagcatcagc caaccaggaa atgattttag 1620cacaaaggat ggagacaacg
acaaatgtat ttgcaaatgt tcacaaatgc taacaggagg 1680ctggtggttt
gatgcatgtg gtccttccaa cttgaacgga atgtactatc cacagaggca
1740gaacacaaat aagttcaacg gcattaaatg gtactactgg aaaggctcag
gctattcgct 1800caaggccaca accatgatga tccgaccagc agatttctaa
acatcccagt ccacctgagg 1860aactgtctcg aactattttc aaagacttaa
gcccagtgca ctgaaagtca cggctgcgca 1920ctgtgtcctc ttccaccaca
gagggcgtgt gctcggtgct gacgggaccc acatgctcca 1980gattagagcc
tgtaaacttt atcacttaaa cttgcatcac ttaacggacc aaagcaagac
2040cctaaacatc cataattgtg attagacaga acacctatgc aaagatgaac
ccgaggctga 2100gaatcagact gacagtttac agacgctgct gtcacaacca
agaatgttat gtgcaagttt 2160atcagtaaat aactggaaaa cagaacactt
atgttataca atacagatca tcttggaact 2220gcattcttct gagcactgtt
tatacactgt gtaaataccc atatgtcct 226910496PRTHomo sapiens 10Met Trp
Gln Ile Val Phe Phe Thr Leu Ser Cys Asp Leu Val Leu Ala1 5 10 15Ala
Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys20 25
30Gln Tyr Gln Val Gln His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro35
40 45Glu Met Asp Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn
Ala50 55 60Val Gln Arg Asp Ala Pro Leu Glu Tyr Asp Asp Ser Val Gln
Arg Leu65 70 75 80Gln Val Leu Glu Asn Ile Met Glu Asn Asn Thr Gln
Trp Leu Met Lys85 90 95Leu Glu Asn Tyr Ile Gln Asp Asn Met Lys Lys
Glu Met Val Glu Ile100 105 110Gln Gln Asn Ala Val Gln Asn Gln Thr
Ala Val Met Ile Glu Ile Gly115 120 125Thr Asn Leu Leu Asn Gln Thr
Ala Glu Gln Thr Arg Lys Leu Thr Asp130 135 140Val Glu Ala Gln Val
Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln Leu145 150 155 160Leu Glu
His Ser Leu Ser Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp165 170
175Gln Thr Ser Glu Ile Asn Lys Leu Gln Asp Lys Asn Ser Phe Leu
Glu180 185 190Lys Lys Val Leu Ala Met Glu Asp Lys His Ile Ile Gln
Leu Gln Ser195 200 205Ile Lys Glu Glu Lys Asp Gln Leu Gln Val Leu
Val Ser Lys Gln Asn210 215 220Ser Ile Ile Glu Glu Leu Glu Lys Lys
Ile Val Thr Ala Thr Val Asn225 230 235 240Asn Ser Val Leu Gln Lys
Gln Gln His Asp Leu Met Glu Thr Val Asn245 250 255Asn Leu Leu Thr
Met Met Ser Thr Ser Asn Ser Ala Lys Asp Pro Thr260 265 270Val Ala
Lys Glu Glu Gln Ile Ser Phe Arg Asp Cys Ala Glu Val Phe275 280
285Lys Ser Gly His Thr Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro
Asn290 295 300Ser Thr Glu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala
Gly Gly Gly305 310 315 320Gly Trp Thr Ile Ile Gln Arg Arg Glu Asp
Gly Ser Val Asp Phe Gln325 330 335Arg Thr Trp Lys Glu Tyr Lys Val
Gly Phe Gly Asn Pro Ser Gly Glu340 345 350Tyr Trp Leu Gly Asn Glu
Phe Val Ser Gln Leu Thr Asn Gln Gln Arg355 360 365Tyr Val Leu Lys
Ile His Leu Lys Asp Trp Glu Gly Asn Glu Ala Tyr370 375 380Ser Leu
Tyr Glu His Phe Tyr Leu Ser Ser Glu Glu Leu Asn Tyr Arg385 390 395
400Ile His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser Ser
Ile405 410 415Ser Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp
Asn Asp Lys420 425 430Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly
Gly Trp Trp Phe Asp435 440 445Ala Cys Gly Pro Ser Asn Leu Asn Gly
Met Tyr Tyr Pro Gln Arg Gln450 455 460Asn Thr Asn Lys Phe Asn Gly
Ile Lys Trp Tyr Tyr Trp Lys Gly Ser465 470 475 480Gly Tyr Ser Leu
Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe485 490
495112424DNAMus musculusmisc_feature(2308)..(2308)n is a, c, g, or
t 11ggctgctcct tcctctcagg acagctccga gtgtgccggg gagaagagaa
gagaagagac 60aggcactggg aaagagcctg ctgcgggacg gagaaggctc tcactgatgg
acttattcac 120acggcacagc cctgtgcctt agacagcagc tgagagctca
ggacgcaagt ttgctgaact 180cacagtttag aacccaaaaa gagagagaga
atgtggcaga tcattttcct aacttttggc 240tgggatcttg tcttggcctc
agcctacagt aactttagga agagcgtgga cagcacaggc 300agaaggcagt
accaggtcca gaacggaccc tgcagctaca cgttcctgct gccggagacc
360gacagctgcc gatcttcctc cagcccctac atgtccaatg ccgtgcagag
ggatgcaccc 420ctcgactacg acgactcagt gcaaaggctg caggtgctgg
agaacattct agagaacaac 480acacagtggc tgatgaagct ggagaattac
attcaggaca acatgaagaa ggagatggtg 540gagatccaac agaatgtggt
gcagaaccag acagctgtga tgatagagat tggaaccagc 600ttgctgaacc
agacagcagc acaaactcgg aaactgactg atgtggaagc ccaagtacta
660aaccagacga caagactcga gctgcagctt ctccaacatt ctatttctac
caacaaattg 720gaaaagcaga ttttggatca gaccagtgaa ataaacaagc
tacaaaataa gaacagcttc 780ctagaacaga aagttctgga catggagggc
aagcacagcg agcagctaca gtccatgaag 840gagcagaagg acgagctcca
ggtgctggtg tccaagcaga gctctgtcat tgacgagctg 900gagaagaagc
tggtgacagc cacggtcaac aactcgctcc ttcagaagca gcagcatgac
960ctaatggaga ccgtcaacag cttgctgacc atgatgtcat cacccaactc
caagagctcg 1020gttgctatcc gtaaagaaga gcaaaccacc ttcagagact
gtgcggaaat cttcaagtca 1080ggactcacca ccagtggcat ctacacactg
accttcccca actccacaga ggagatcaag 1140gcctactgtg acatggacgt
gggtggagga gggtggacag tcatccaaca ccgagaagat 1200ggcagtgtgg
acttccagag gacgtggaaa gaatacaaag agggcttcgg gaaccctctg
1260ggagagtact ggctgggcaa tgagtttgtc tcccagctga ccggtcagca
ccgctacgtg 1320cttaagatcc agctgaagga ctgggaaggc aacgaggcgc
attcgctgta tgatcacttc 1380tacctcgctg gtgaagagtc caactacagg
attcacctta caggactcac ggggaccgcg 1440gccaaaataa gtagcatcag
ccaaccagga agtgatttta gcacaaagga ttcggacaat 1500gacaaatgca
tctgcaagtg ttcccagatg ctctcaggag gctggtggtt tgacgcatgt
1560ggtccttcca acttgaatgg acagtactac ccacaaaaac agaatacaaa
taagtttaac 1620ggtatcaagt ggtactactg gaaggggtcc ggctactcgc
tcaaggccac aaccatgatg 1680atccggccag cagatttcta aatgcctgcc
tacactacca gaagaacttg ctgcatccaa 1740agattaactc caaggcactg
agagacacca gtgcatagca gcccctttcc acatcaggaa 1800gtgctcctgg
gggtggggag ggtctgtgtg taccagactg aagcgcatca cttaagcctg
1860caccgctaac caaccaaagg cactgcagtc tggagaaaca cttctgggaa
ggttgtggct 1920gaggatcaga aggacagcgt gcagactctg tcacaaggaa
gaatgttccg tgggagttca 1980gcagtaaata actggaaaac agaacactta
gatggtgcag ataaatcttg ggaccacatt 2040cctctaagca cggtttctag
agtgaataca ttcacagctc ggctgtcaca atgacaaggc 2100cgtgtcctcg
cactgtggca gccagtatcc agggacttct aagtggtggg cacaggctat
2160catctggaga agcacacatt cattgttttc ctcttgggtg cttaacatgt
tcatttgaaa 2220acaacacatt tacctatctt gatggcttag tttttaatgg
ctggctacta tttactatat 2280ggcaaaaatg cccacatctc tggaatancc
accaaataag cgccatgttg gtgaatgcgg 2340aggctgtact attttgtttt
cttcctggct ggtaaatatg aaggtatttt tagtaattaa 2400atataagtta
ttagttgaaa gacc 242412496PRTMus musculus 12Met Trp Gln Ile Ile Phe
Leu Thr Phe Gly Trp Asp Leu Val Leu Ala1 5 10 15Ser Ala Tyr Ser Asn
Phe Arg Lys Ser Val Asp Ser Thr Gly Arg Arg20 25 30Gln Tyr Gln Val
Gln Asn Gly Pro Cys Ser Tyr Thr Phe Leu Leu Pro35 40 45Glu Thr Asp
Ser Cys Arg Ser Ser Ser Ser Pro Tyr Met Ser Asn Ala50 55 60Val Gln
Arg Asp Ala Pro Leu Asp Tyr Asp Asp Ser Val Gln Arg Leu65 70 75
80Gln Val Leu Glu Asn Ile Leu Glu Asn Asn Thr Gln Trp Leu Met Lys85
90 95Leu Glu Asn Tyr Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu
Ile100 105 110Gln Gln Asn Val Val Gln Asn Gln Thr Ala Val Met Ile
Glu Ile Gly115 120 125Thr Ser Leu Leu Asn Gln Thr Ala Ala Gln Thr
Arg Lys Leu Thr Asp130 135 140Val Glu Ala Gln Val Leu Asn Gln Thr
Thr Arg Leu Glu Leu Gln Leu145 150 155 160Leu Gln His Ser Ile Ser
Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp165 170 175Gln Thr Ser Glu
Ile Asn Lys Leu Gln Asn Lys Asn Ser Phe Leu Glu180 185 190Gln Lys
Val Leu Asp Met Glu Gly Lys His Ser Glu Gln Leu Gln Ser195 200
205Met Lys Glu Gln Lys Asp Glu Leu Gln Val Leu Val Ser Lys Gln
Ser210 215 220Ser Val Ile Asp Glu Leu Glu Lys Lys Leu Val Thr Ala
Thr Val Asn225 230 235 240Asn Ser Leu Leu Gln Lys Gln Gln His Asp
Leu Met Glu Thr Val Asn245 250 255Ser Leu Leu Thr Met Met Ser Ser
Pro Asn Ser Lys Ser Ser Val Ala260 265 270Ile Arg Lys Glu Glu Gln
Thr Thr Phe Arg Asp Cys Ala Glu Ile Phe275 280 285Lys Ser Gly Leu
Thr Thr Ser Gly Ile Tyr Thr Leu Thr Phe Pro Asn290 295 300Ser Thr
Glu Glu Ile Lys Ala Tyr Cys Asp Met Asp Val Gly Gly Gly305 310 315
320Gly Trp Thr Val Ile Gln His Arg Glu Asp Gly Ser Val Asp Phe
Gln325 330 335Arg Thr Trp Lys Glu Tyr Lys Glu Gly Phe Gly Asn Pro
Leu Gly Glu340 345 350Tyr Trp Leu Gly Asn Glu Phe Val Ser Gln Leu
Thr Gly Gln His Arg355 360 365Tyr Val Leu Lys Ile Gln Leu Lys Asp
Trp Glu Gly Asn Glu Ala His370 375 380Ser Leu Tyr Asp His Phe Tyr
Leu Ala Gly Glu Glu Ser Asn Tyr Arg385 390 395 400Ile His Leu Thr
Gly Leu Thr Gly Thr Ala Ala Lys Ile Ser Ser Ile405 410 415Ser Gln
Pro Gly Ser Asp Phe Ser Thr Lys Asp Ser Asp Asn Asp Lys420 425
430Cys Ile Cys Lys Cys Ser Gln Met Leu Ser Gly Gly Trp Trp Phe
Asp435 440 445Ala Cys Gly Pro Ser Asn Leu Asn Gly Gln Tyr Tyr Pro
Gln Lys Gln450 455 460Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr
Tyr Trp Lys Gly Ser465 470 475 480Gly Tyr Ser Leu Lys Ala Thr Thr
Met Met Ile Arg Pro Ala Asp Phe485 490 4951324DNAPrimer Sequence
13acaatgacag ttttcctttc cttt 241424DNAPrimer Sequence 14tgtgtccatg
agctccagtt gttg 241524DNAPrimer Sequence 15aaatggtttc tcttcttctc
tttt 241624DNAPrimer Sequence 16cgagactgtg cagatgtata tcaa
241724DNAPrimer Sequence 17tcttctccct ccgttttctg gatt
241824DNAPrimer Sequence 18agtatggact ctttagccgg ctta
241924DNAPrimer Sequence 19catctttccc cctccgaggt ctgc 24
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